Cyclic di-nucleotide compounds as sting agonists

ABSTRACT

A class of polycyclic compounds of general formula (I), of general formula (I′), or of general formula (I″), wherein Base 1 , Base 2 , Y, Y a , X a , X a1 , X b , X b1 , X c , X c1 , X d , X d1 , R 1 , R 1a , R 2 , R 2a , R 3 , R 4 , R 4a , R 5 , R 6 , R 6a , R 7 , R 7a , R 8 , and R 8a  are defined herein, that may be useful as inductors of type I interferon production, specifically as STING active agents, are provided. Also provided are processes for the synthesis and use of compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/204,677, filed Aug. 13, 2015, U.S. Provisional PatentApplication No. 62/268,723, filed Dec. 17, 2015, and U.S. ProvisionalPatent Application No. 62/356,125, filed Jun. 29, 2016.

FIELD OF THE INVENTION

The present disclosure relates to cyclic di-nucleotide compounds andderivatives thereof that may be useful as STING (Stimulator ofInterferon Genes) agonists that activate the STING pathway. The presentdisclosure also relates to processes for the synthesis and to uses ofsuch cyclic di-nucleotide compounds.

BACKGROUND OF THE INVENTION

The immune system has evolved to recognize and neutralize differenttypes of threats in order to maintain the homeostasis of the host, andit is generally broken down into two arms: adaptive and innate. Theadaptive immune system is specialized to recognize antigens notnaturally expressed in the host as foreign and to mount an anti-antigenresponse through the coordinated actions of many leukocyte subsets. Thehallmark of adaptive immune responses is their ability to provide“memory” or long-lasting immunity against the encountered antigen. Whilethis specific and long-lasting effect is critical to host health andsurvival, the adaptive immune response requires time to generate afull-blown response.

The innate immune system compensates for this time delay and isspecialized to act quickly against different insults or danger signals.It provides the first line of defense against bacteria, viruses,parasites and other infectious threats, but it also responds strongly tocertain danger signals associated with cellular or tissue damage. Theinnate immune system has no antigen specificity but does respond to avariety of effector mechanisms. Opsonization, phagocytosis, activationof the complement system, and production of soluble bioactive moleculessuch as cytokines or chemokines are all mechanisms by which the innateimmune system mediates its response. By responding to thesedamage-associated molecular patterns (DAMPs) or pathogen-associatedmolecular patterns (PAMPs) described above, the innate immune system isable to provide broad protection against a wide range of threats to thehost.

Free cytosolic DNA and RNA are among these PAMPs and DAMPs. It hasrecently been demonstrated that the main sensor for cytosolic DNA iscGAS (cyclic GMP-AMP synthase). Upon recognition of cytosolic DNA, cGAScatalyzes the generation of the cyclic-dinucleotide 2′-3′ cGAMP, anatypical second messenger that strongly binds to the ER-transmembraneadaptor protein STING. A conformational change is undergone bycGAMP-bound STING, which translocates to a perinuclear compartment andinduces the activation of critical transcription factors IRF-3 andNF-κB. This leads to a strong induction of type I interferons andproduction of pro-inflammatory cytokines such as IL-6, TNF-α and IFN-γ.

The importance of type I interferons and pro-inflammatory cytokines onvarious cells of the immune system has been very well established. Inparticular, these molecules strongly potentiate T cell activation byenhancing the ability of dendritic cells and macrophages to uptake,process, present and cross-present antigens to T cells. The T cellstimulatory capacity of these antigen-presenting cells is augmented bythe up-regulation of critical co-stimulatory molecules, such as CD80 orCD86. Finally, type I interferons can rapidly engage their cognatereceptors and trigger the activation of interferon-responsive genes thatcan significantly contribute to adaptive immune cell activation.

From a therapeutic perspective, type I interferons are shown to haveantiviral activities by directly inhibiting human hepatitis B virus andhepatitis C virus replication, and by stimulating immune responses tovirally infected cells. Compounds that can induce type I interferonproduction are used in vaccines, where they act as adjuvants, enhancingspecific immune responses to antigens and minimizing side effects byreducing dosage and broadening the immune response.

In addition, interferons, and compounds that can induce interferonproduction, have potential use in the treatment of human cancers. Suchmolecules are potentially useful as anti-cancer agents with multiplepathways of activity. Interferons can inhibit human tumor cellproliferation directly and may be synergistic with various approvedchemotherapeutic agents. Type I interferons can significantly enhanceanti-tumor immune responses by inducing activation of both the adaptiveand innate immune cells. Finally, tumor invasiveness may be inhibited byinterferons by modulating enzyme expression related to tissueremodeling.

In view of the potential of type I interferons and type Iinterferon-inducing compounds as anti-viral and anti-cancer agents,there remains a need for new agents that can induce potent type Iinterferon production. With the growing body of data demonstrating thatthe cGAS-STING cytosolic DNA sensory pathway has a significant capacityto induce type I interferons, the development of STING activating agentsis rapidly taking an important place in today's anti-tumor therapylandscape.

SUMMARY OF THE INVENTION

The present disclosure relates to novel cyclic di-nucleotide compoundsof general formula (I), general formula (I′), and/or general formula(I″). In particular, the present disclosure relates to compounds havingthe general structural formula (I):

or pharmaceutically acceptable salts, hydrates, solvates, or prodrugsthereof, as described herein. The present disclosure also relates tocompounds having general structural formula (I′):

or pharmaceutically acceptable salts, hydrates, solvates, or prodrugsthereof, as described herein. The present disclosure also relates tocompounds having general structural formula (I″):

or pharmaceutically acceptable salts, hydrates, solvates, or prodrugsthereof, as described herein.

Embodiments of the disclosure include compounds of general formula (I),compounds of general formula (I′), and/or compounds of general formula(I″), and pharmaceutically acceptable salts, hydrates, solvates, orprodrugs thereof, as well as synthesis and isolation of compounds ofgeneral formula (I), compounds of general formula (I′), and/or compoundsof general formula (I″), and pharmaceutically acceptable salts,hydrates, solvates, or prodrugs thereof. Uses of compounds of generalformula (I), compounds of general formula (I′), and/or compounds ofgeneral formula (I″) are also disclosed.

Other embodiments, aspects and features of the present disclosure areeither further described in or will be apparent from the ensuingdescription, examples and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Not Applicable

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure includes compounds of general formula (I),compounds of general formula (I′), and/or compounds of general formula(I″) above, and pharmaceutically acceptable salts, hydrates, solvates,or prodrugs thereof. These compounds and their pharmaceuticallyacceptable salts, hydrates, solvates, and/or prodrugs are useful asagents to induce interferon production.

A first embodiment of the disclosure relates to cyclic di-nucleotidecompounds of general formula (I):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, wherein Base¹ and Base² are each independently selected fromthe group consisting of

where Base¹ and Base² each may be independently substituted by 0-3substituents R¹⁰, where each R¹⁰ is independently selected from thegroup consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, andN(C₃₋₆ cycloalkyl)₂; Y and Y^(a) are each independently selected fromthe group consisting of —O—, —S—, —SO₂—, —CH₂—, and —CF₂—; X^(a) andX^(a1) are each independently selected from the group consisting of O,C, and S; X^(b) and X^(b1) are each independently selected from thegroup consisting of O, C, and S; X^(c) and X^(c1) are each independentlyselected from the group consisting of O⁻, S⁻, OR⁹, and NR⁹R⁹; X^(d) andX^(d1) are each independently selected from the group consisting of Oand S; R¹ and R^(1a) are each independently selected from the groupconsisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl,—O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R¹and R^(1a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl,C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl,and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selectedfrom the group consisting of F, Cl, Br, I, OH, CN, and N₃; R² and R^(2a)are each independently selected from the group consisting of H, F, Cl,Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R² and R^(2a) C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl,C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆alkynyl are substituted by 0 to 3 substituents selected from the groupconsisting of F, Cl, Br, I, OH, CN, and N₃; R³ is selected from thegroup consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl,where said R³ C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3substituents selected from the group consisting of F, Cl, Br, I, OH, CN,and N₃; R⁴ and R^(4a) are each independently selected from the groupconsisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl,—O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁴and R^(4a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl,C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl,and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selectedfrom the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁵ isselected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl,C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆alkynyl, where said R⁵ C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3substituents selected from the group consisting of F, Cl, Br, I, OH, CN,and N₃; R⁶ and R^(6a) are each independently selected from the groupconsisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl,—O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁶and R^(6a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl,C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl,and —O—C₂-C₆ alkynyl are substituted by 0 to 3 substituents selectedfrom the group consisting of F, Cl, Br, I, OH, CN, and N₃; R⁷ and R^(7a)are each independently selected from the group consisting of H, F, Cl,Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁷ and R^(7a) C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl,C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆alkynyl are substituted by 0 to 3 substituents selected from the groupconsisting of F, Cl, Br, I, OH, CN, and N₃; R⁸ and R^(8a) are eachindependently selected from the group consisting of H, F, Cl, Br, I, OH,CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl,C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl,and —O—C₂-C₆ alkynyl, where said R⁸ and R^(8a) C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, Br, I, OH, CN, and N₃; each R⁹ is independently selected from thegroup consisting of H, C₁-C₂₀ alkyl,

where each R⁹ C₁-C₂₀ alkyl is optionally substituted by 0 to 3substituents independently selected from the group consisting of OH,—O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; optionallyR^(1a) and R³ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene,C₂-C₆ alkynylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆alkynylene, such that where R^(1a) and R³ are connected to form —O—C₁-C₆alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is boundat the R³ position; optionally R^(2a) and R³ are connected to form C₁-C₆alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene, —O—C₁-C₆ alkylene,—O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R^(2a) andR³ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or—O—C₂-C₆ alkynylene, said O is bound at the R³ position; optionally R³and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene,or —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected toform —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene,said O is bound at the R³ position; optionally R⁴ and R⁵ are connectedto form are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆alkynylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆alkynylene, such that where R⁴ and R⁵ are connected to form —O—C₁-C₆alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is boundat the R⁵ position; optionally R⁵ and R⁶ are connected to form —O—C₁-C₆alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that whereR⁵ and R⁶ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene,or —O—C₂-C₆ alkynylene, said O is bound at the R⁵ position; optionallyR⁷ and R⁸ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, orC₂-C₆ alkynylene; and optionally R^(7a) and R^(8a) are connected to formC₁-C₆ alkylene, C₂-C₆ alkenylene, or C₂-C₆ alkynylene.

In specific aspects of this embodiment, when Y and Y^(a) are each O,X^(a) and X^(a1) are each O, X^(b) and X^(b1) are each O, and X^(c) andX^(c1) are each OH or SH, X^(d) and X^(d1) are each O, R¹ and R^(1a) areeach H, R² is H, R⁶ and R^(6a) are each H, R⁷ and R^(7a) are each H, R⁸and R^(8a) are each H, and Base¹ and Base² are each selected from thegroup consisting of

R⁵ and R³ are not both selected from the group consisting of H, F andOH. That is, when Y and Y^(a) are each O, X^(a) and X^(a1) are each O,X^(b) and X^(b1) are each O, and X^(c) and X^(c1) are each OH or SH,X^(d) and X^(d1) are each O, R¹ and R^(1a) are each H, R² is H, R⁶ andR^(6a) are each H, R⁷ and R^(7a) are each H, R⁸ and R^(8a) are each H,and Base¹ and Base² are each selected from the group consisting of

either only one of R⁵ and R³ is selected from the group consisting of H,F, and OH, or neither R⁵ and R³ is selected from the group consisting ofH, F, and OH. In further specific instances of this aspect, when Y andY^(a) are each O, X^(a) and X^(a1) are each O, X^(b) and X^(b1) are eachO, and X^(c) and X^(c1) are each OH, X^(d) and X^(d1) are each O or S,R¹ and R^(1a) are each H, R² is H, R⁶ and R^(6a) are each H, R⁷ andR^(7a) are each H, R⁸ and R^(8a) are each H, and Base¹ and Base² areeach selected from the group consisting of

R⁵ and R³ are not both selected from the group consisting of H, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, where said C₁-C₆ alkyl, C₂-C₆alkenyl and C₂-C₆ alkynyl are substituted by 0 to 3 substituentsselected from the group consisting of F, Cl, Br, I and OH.

In further specific aspects of this embodiment, when Base¹ and Base² areeach selected from the group consisting of

and R^(2a) is F and R⁵ is F, at least one of X^(c) and X^(c1) is SR⁹.

In a first aspect of the first embodiment, Base¹ and Base² are eachindependently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3substituents R¹⁰, where each R¹⁰ is independently selected from thegroup consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, andN(C₃₋₆ cycloalkyl)₂. In particular instances, Base¹ and Base² are eachindependently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3substituents R¹⁰, where each R¹⁰ is independently selected from thegroup consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, andN(C₃₋₆ cycloalkyl)₂. In even more particular instances, Base¹ and Base²are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3substituents R¹⁰, where each R¹⁰ is independently selected from thegroup consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, andN(C₃₋₆ cycloalkyl)₂. In this aspect, all other groups are as provided inthe general formula (I) of the first embodiment above.

In a second aspect of the first embodiment, Y and Y^(a) are eachindependently selected from the group consisting of —O— and —S—. In thisaspect, all other groups are as provided in the general formula (I) ofthe first embodiment above or in the first aspect described above.

In a third aspect of the first embodiment, X^(a) and X^(a1) are eachindependently selected from the group consisting of O and S. In thisaspect, all other groups are as provided in the general formula (I) ofthe first embodiment above or in the first through second aspectsdescribed above.

In a fourth aspect of the first embodiment, X^(b) and X^(b1) are eachindependently selected from the group consisting of O and S. In thisaspect, all other groups are as provided in the general formula (I) ofthe first embodiment above or in the first through third aspectsdescribed above.

In a fifth aspect of the first embodiment, X^(c) and X^(c1) are eachindependently selected from the group consisting of O⁻, S⁻, OR⁹, andNR⁹R⁹, where each R⁹ is independently selected from the group consistingof H, C₁-C₂₀ alkyl,

where each R⁹ C₁-C₂₀ alkyl is optionally substituted by 0 to 3substituents independently selected from the group consisting of OH,—O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl. In particularinstances, X^(c) and X^(c) are each independently selected from thegroup consisting of O⁻, S⁻,

In all instances of this aspect, all other groups are as provided in thegeneral formula (I) of the first embodiment above or in the firstthrough fourth aspects described above.

In a sixth aspect of the first embodiment, X^(d) and X^(d1) are eachindependently selected from the group consisting of O and S. In thisaspect, all other groups are as provided in the general formula (I) ofthe first embodiment above or in the first through fifth aspectsdescribed above.

In a seventh aspect of the first embodiment, R¹ and R^(1a) are each H.In this aspect, all other groups are as provided in the general formula(I) of the first embodiment above or in the first through sixth aspectsdescribed above.

In an eighth aspect of the first embodiment, R² and R^(2a) are eachindependently selected from the group consisting of H, F, Cl, I, Br, OH,N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R² and R^(2a) C₁-C₆alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selectedfrom the group consisting of F, Cl, Br, I, OH, CN, and N₃. In particularinstances, R² and R^(2a) are each independently selected from the groupconsisting of H, F, Cl, I, Br, OH, CN, N₃, CF₃, CH₃, CH₂OH, and CH₂CH₃.In this aspect, all other groups are as provided in the general formula(I) of the first embodiment above or in the first through seventhaspects described above.

In a ninth aspect of the first embodiment, R³ is selected from the groupconsisting H, F, Cl, I, Br, OH, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl,where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3substituents selected from the group consisting of F, Cl, Br, I, OH, CN,and N₃. In particular instances, R³ are each independently selected fromthe group consisting of H, F, Cl, I, Br, OH, CN, N₃, CF₃, CH₃, CH₂OH,and CH₂CH₃. In this aspect, all other groups are as provided in thegeneral formula (I) of the first embodiment above or in the firstthrough eighth aspects described above.

In a tenth aspect of the first embodiment, R⁴ and R^(4a) are eachindependently selected from the group consisting of H, F, Cl, I, Br, OH,N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁴ and R^(4a) C₁-C₆alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selectedfrom the group consisting of F, Cl, Br, I, OH, CN, and N₃. In particularinstances, R⁴ and R^(4a) are each independently selected from the groupconsisting of H, F, Cl, I, Br, OH, CN, N₃, CF₃, CH₃, CH₂OH, and CH₂CH₃.In this aspect, all other groups are as provided in the general formula(I) of the first embodiment above or in the first through ninth aspectsdescribed above.

In an eleventh aspect of the first embodiment, R⁵ is selected from thegroup consisting of H, F, Cl, I, Br, OH, N₃, C₁-C₆ alkyl, and C₁-C₆haloalkyl, where said R⁵ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substitutedby 0 to 3 substituents selected from the group consisting of F, Cl, Br,I, OH, CN, and N₃. In particular instances, R⁵ are each independentlyselected from the group consisting of H, F, Cl, I, Br, OH, CN, N₃, CF₃,CH₃, CH₂OH, and CH₂CH₃. In this aspect, all other groups are as providedin the general formula (I) of the first embodiment above or in the firstthrough tenth aspects described above.

In a twelfth aspect of the first embodiment, R⁶ and R^(6a) are eachindependently selected from the group consisting of H, F, Cl, I, Br, OH,C₁-C₆ alkyl, C₂-C₆ alkenyl, and C₂-C₆ alkynyl. In this aspect, all othergroups are as provided in the general formula (I) of the firstembodiment above or in the first through eleventh aspects describedabove.

In a thirteenth aspect of the first embodiment, R⁷ and R^(7a) are eachH. In this aspect, all other groups are as provided in the generalformula (I) of the first embodiment above or in the first throughtwelfth aspects described above.

In a fourteenth aspect of the first embodiment, R⁸ and R^(8a) are eachH. In this aspect, all other groups are as provided in the generalformula (I) of the first embodiment above or in the first throughthirteenth aspects described above.

In a fifteenth aspect of the first embodiment, R^(1a) and R³ areconnected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene,—O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, suchthat where R^(1a) and R³ are connected to form —O—C₁-C₆ alkylene,—O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³position. In this aspect, all other groups are as provided in thegeneral formula (I) of the first embodiment above or in the firstthrough fourteenth aspects described above.

In a sixteenth aspect of the first embodiment, R^(2a) and R³ areconnected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene,—O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, suchthat where R^(2a) and R³ are connected to form —O—C₁-C₆ alkylene,—O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³position. In this aspect, all other groups are as provided in thegeneral formula (I) of the first embodiment above or in the firstthrough fourteenth aspects described above.

In a seventeenth aspect of the first embodiment, R³ and R^(6a) areconnected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, and —O—C₂-C₆alkynylene, such that where R³ and R^(6a) are connected to form —O—C₁-C₆alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is boundat the R³ position. In this aspect, all other groups are as provided inthe general formula (I) of the first embodiment above or in the firstthrough fourteenth aspects described above.

In an eighteenth aspect of the first embodiment, R⁴ and R⁵ are connectedby C₁-C₆ alkylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆alkynylene, such that where R⁴ and R⁵ are connected to form —O—C₁-C₆alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is boundat the R⁵ position. In this aspect, all other groups are as provided inthe general formula (I) of the first embodiment above or in the firstthrough fourteenth aspects described above.

In a nineteenth aspect of the first embodiment, R⁵ and R⁶ are connectedto form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene,such that where R⁵ and R⁶ are connected to form —O—C₁-C₆ alkylene,—O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R⁵position. In this aspect, all other groups are as provided in thegeneral formula (I) of the first embodiment above or in the firstthrough fourteenth aspects described above.

In a twentieth aspect of the first embodiment, R⁷ and R⁸ are connectedto form C₁-C₆ alkylene, C₂-C₆ alkenylene, or C₂-C₆ alkynylene. In thisaspect, all other groups are as provided in the general formula (I) ofthe first embodiment above or in the first through fourteenth aspectsdescribed above.

In a twenty-first aspect of the first embodiment, R^(7a) and R^(8a) areconnected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, or C₂-C₆ alkynylene.In this aspect, all other groups are as provided in the general formula(I) of the first embodiment above or in the first through fourteenthaspects described above.

In a twenty-second aspect of the first embodiment, Base¹ and Base² areeach independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3substituents R¹⁰, where each R¹⁰ is independently selected from thegroup consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, andN(C₃₋₆ cycloalkyl)₂; Y and Y^(a) are each independently selected fromthe group consisting of —O—, —S—, —SO₂—, —CH₂—, and —CF₂—; X^(a) andX^(a1) are each independently selected from the group consisting of Oand S; X^(b) and X^(b1) are each independently selected from the groupconsisting of O and S; X^(c) and X^(c1) are each independently selectedfrom the group consisting of O⁻, S⁻, OR⁹, and NR⁹R⁹; X^(d) and X^(d1)are each independently selected from the group consisting of O and S; R¹and R^(1a) are each H; R² and R^(2a) are each independently selectedfrom the group consisting of H, F, Cl, I, Br, OH, N₃, C₁-C₆ alkyl, andC₁-C₆ haloalkyl, where said R² and R^(2a) C₁-C₆ alkyl or C₁-C₆ haloalkylare substituted by 0 to 3 substituents selected from the groupconsisting of F, Cl, Br, I, OH, CN, and N₃; R³ is selected from thegroup consisting of H, F, Cl, I, Br, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substitutedby 0 to 3 substituents selected from the group consisting of F, Cl, Br,I, OH, CN, and N₃; R⁴ and R^(4a) are each independently selected fromthe group consisting of H, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where saidR⁴ and R^(4a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3substituents selected from the group consisting of F, Cl, Br, I, OH, CN,and N₃; R⁵ is selected from the group consisting of H, F, Cl, I, Br, OH,N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁵ C₁-C₆ alkyl or C₁-C₆haloalkyl are substituted by 0 to 3 substituents selected from the groupconsisting of F, Cl, Br, I, OH, CN, and N₃; R⁶ and R^(6a) are eachindependently selected from the group consisting of H, F, Cl, I, Br, OH,C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, and C₁-C₆ haloalkyl, wheresaid R⁶ and R^(6a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0to 3 substituents selected from the group consisting of F, Cl, Br, I,OH, CN, and N₃; R⁷ and R^(7a) are each H; R⁸ and R^(8a) are each H; eachR⁹ is independently selected from the group consisting of H, C₂-C₃alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2substituents independently selected from the group consisting of OH,—O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; optionally R³and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene,and —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected toform —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene,said O is bound at the R³ position or optionally R⁴ and R⁵ are connectedby C₁-C₆ alkylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆alkynylene, such that where R⁴ and R⁵ are connected to form —O—C₁-C₆alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is boundat the R⁵ position. In this aspect, all other groups are as provided inthe general formula (I) of the first embodiment above.

In a twenty-third aspect of the first embodiment, the compound offormula (I) is a compound of formula (Ia):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, wherein Base¹ and Base² are each independently selected fromthe group consisting of

where Base¹ and Base² each may be independently substituted by 0-3substituents R¹⁰, where each R¹⁰ is independently selected from thegroup consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, andN(C₃₋₆ cycloalkyl)₂; X^(c) and X^(c1) are each independently selectedfrom the group consisting of O⁻, S⁻, OR⁹, and NR⁹R⁹; R³ is selected fromthe group consisting of H, F, Cl, I, Br, OH, CN, N₃, C₁-C₆ alkyl, andC₁-C₆ haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, I, Br, and OH; R⁵ is selected from the group consisting of H, F,Cl, I, Br, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁵C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituentsselected from the group consisting of F, Cl, I, Br, and OH; R³ and R⁵are not both selected from the group consisting of OH, C₁-C₆ alkylsubstituted with OH, and C₁-C₆ haloalkyl substituted with OH; and eachR⁹ is independently selected from the group consisting of H, C₂-C₃alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2substituents independently selected from the group consisting of OH,—O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl. In thisaspect, all other groups are as provided in the general formula (I) ofthe first embodiment above.

In a twenty-fourth aspect of the first embodiment, the compound offormula (I) is a compound of formula (Ib):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, wherein Base¹ and Base² are each independently selected fromthe group consisting of

where Base¹ and Base² each may be independently substituted by 0-3substituents R¹⁰, where each R¹⁰ is independently selected from thegroup consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, andN(C₃₋₆ cycloalkyl)₂; X^(c) and X^(c1) are each independently selectedfrom the group consisting of O⁻, S⁻, OR⁹, and NR⁹R⁹; R^(1a) is selectedfrom the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl,where said R^(1a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3substituents selected from the group consisting of F, Cl, Br, I, OH, CN,and N₃; R^(2a) is selected from the group consisting of H, F, Cl, I, Br,OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R^(2a) C₁-C₆alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selectedfrom the group consisting of F, Cl, I, Br, and OH; R³ is selected fromthe group consisting of H, F, Cl, I, Br, OH, CN, N₃, C₁-C₆ alkyl, andC₁-C₆ haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, I, Br, and OH; R⁵ is selected from the group consisting of H, F,Cl, I, Br, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁵C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituentsselected from the group consisting of F, Cl, I, Br, and OH; R³ and R⁵are not both selected from the group consisting of OH, C₁-C₆ alkylsubstituted with OH, and C₁-C₆ haloalkyl substituted with OH; R^(6a) isselected from the group consisting of H, F, Cl, I, Br, OH, C₁-C₆ alkyl,C₂-C₆ alkenyl, and C₂-C₆ alkynyl; each R⁹ is independently selected fromthe group consisting of H, C₂-C₃ alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2substituents independently selected from the group consisting of OH,—O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; andoptionally R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene,—O—C₂-C₆ alkenylene, and —O—C₂-C₆ alkynylene, such that where R³ andR^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or—O—C₂-C₆ alkynylene, said O is bound at the R³ position. In this aspect,all other groups are as provided in the general formula (I) of the firstembodiment above.

In a twenty-fifth aspect of the first embodiment, the compound offormula (I) is a compound of formula (Ic):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, wherein Base¹ and Base² are each independently selected fromthe group consisting of

where Base¹ and Base² each may be independently substituted by 0-3substituents R¹⁰, where each R¹⁰ is independently selected from thegroup consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, andN(C₃₋₆ cycloalkyl)₂; X^(c) and X^(c1) are each independently selectedfrom the group consisting of O⁻, S⁻, OR⁹, and NR⁹R⁹; R³ is selected fromthe group consisting of H, F, Cl, I, Br, OH, CN, N₃, C₁-C₆ alkyl, andC₁-C₆ haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, I, Br, and OH; R⁴ is selected from the group consisting of H, F,OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁴ C₁-C₆ alkylor C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected fromthe group consisting of F, Cl, I, Br, and OH; R⁵ is selected from thegroup consisting of H, F, Cl, I, Br, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆haloalkyl, where said R⁵ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substitutedby 0 to 3 substituents selected from the group consisting of F, Cl, I,Br, and OH; R³ and R⁵ are not both selected from the group consisting ofOH, C₁-C₆ alkyl substituted with OH, and C₁-C₆ haloalkyl substitutedwith OH; R^(6a) is selected from the group consisting of H, F, Cl, I,Br, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R^(6a)C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituentsselected from the group consisting of F, Cl, I, Br, and OH; each R⁹ isindependently selected from the group consisting of H, C₂-C₃ alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2substituents independently selected from the group consisting of OH,—O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; andoptionally R⁴ and R⁵ are connected by C₁-C₆ alkylene, —O—C₁-C₆ alkylene,—O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R⁴ and R⁵are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or—O—C₂-C₆ alkynylene, said O is bound at the R⁵ position. In this aspect,all other groups are as provided in the general formula (I) of the firstembodiment above.

A twenty-sixth aspect of the first embodiment relates to apharmaceutical composition, said pharmaceutical composition comprising(a) a compound according to any one of general formula (I) of the firstembodiment above or in the first through twenty-fifth aspects describedabove or a pharmaceutically acceptable salt, hydrate, solvate, orprodrug thereof, and (b) a pharmaceutically acceptable carrier.

A twenty-seventh aspect of the first embodiment relates to methods ofinducing an immune response in a subject, comprising administering atherapeutically effective amount of a compound according to any one ofgeneral formula (I) of the first embodiment above or in the firstthrough twenty-fifth aspects described above or a pharmaceuticallyacceptable salt, hydrate, solvate, or prodrug thereof to the subject.

A twenty-eighth aspect of the first embodiment relates to methods ofinducing an immune response in a subject, comprising administering atherapeutically effective amount of a composition according to thetwenty-sixth aspect described above to the subject.

A twenty-ninth aspect of the first embodiment relates to methods ofinducing a STING-dependent type I interferon production in a subject,comprising administering a therapeutically effective amount of acompound according to any one of general formula (I) of the firstembodiment above or in the first through twenty-fifth aspects describedabove or a pharmaceutically acceptable salt, hydrate, solvate, orprodrug thereof to the subject.

A thirtieth aspect of the first embodiment relates to methods ofinducing a STING-dependent type I interferon production in a subject,comprising administering a therapeutically effective amount of acomposition according to the twenty-sixth aspect described above to thesubject.

A thirty-first aspect of the first embodiment relates to methods ofinducing a STING-dependent cytokine production in a subject, comprisingadministering a therapeutically effective amount of a compound accordingto any one of general formula (I) of the first embodiment above or inthe first through twenty-fifth aspects described above or apharmaceutically acceptable salt, hydrate, solvate, or prodrug thereofto the subject.

A thirty-second aspect of the first embodiment relates to methods ofinducing a STING-dependent cytokine production in a subject, comprisingadministering a therapeutically effective amount of a compositionaccording to the twenty-sixth aspect described above to the subject.

A second embodiment of the disclosure relates to cyclic di-nucleotidecompounds of general formula (I′):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, wherein Base¹ and Base² are each independently selected fromthe group consisting of

where Base and Base² each may be independently substituted by 0-3substituents R¹⁰, where each R¹⁰ is independently selected from thegroup consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃-6cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, andN(C₃₋₆ cycloalkyl)₂; Y and Y^(a) are each independently selected fromthe group consisting of —O— and —S—; X^(a) and X^(a1) are eachindependently selected from the group consisting of O, and S; X^(b) andX^(b1) are each independently selected from the group consisting of O,and S; X^(c) and X^(c1) are each independently selected from the groupconsisting of OR⁹, SR⁹, and NR⁹R⁹; X^(d) and X^(c1) are eachindependently selected from the group consisting of O and S; R¹ andR^(1a) are each independently selected from the group consisting of H,F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R¹ and R^(1a) C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl,C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆alkynyl are substituted by 0 to 3 substituents selected from the groupconsisting of F, Cl, Br, I, OH, CN, and N₃; R² and R^(2a) are eachindependently selected from the group consisting of H, F, Cl, Br, I, OH,CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl,C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl,and —O—C₂-C₆ alkynyl, where said R² and R^(2a) C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, Br, I, OH, CN, and N₃; R³ is selected from the group consistingof H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R³ C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl,C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆alkynyl are substituted by 0 to 3 substituents selected from the groupconsisting of F, Cl, Br, I, OH, CN, and N₃; R⁴ and R^(4a) are eachindependently selected from the group consisting of H, F, Cl, Br, I, OH,CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl,C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl,and —O—C₂-C₆ alkynyl, where said R⁴ and R^(4a) C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, Br, I, OH, CN, and N₃; R⁵ is selected from the group consistingof H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁵ C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl,C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆alkynyl are substituted by 0 to 3 substituents selected from the groupconsisting of F, Cl, Br, I, OH, CN, NR⁹R⁹, and N₃; R⁶ and R^(6a) areeach independently selected from the group consisting of H, F, Cl, Br,I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁶ and R^(6a) C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl,C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆alkynyl are substituted by 0 to 3 substituents selected from the groupconsisting of F, Cl, Br, I, OH, CN, and N₃; R⁷ and R^(7a) are eachindependently selected from the group consisting of H, F, Cl, Br, I, OH,CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl,C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl,and —O—C₂-C₆ alkynyl, where said R⁷ and R^(7a) C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, Br, I, OH, CN, and N₃; R⁸ and R^(8a) are each independentlyselected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl,C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆alkynyl, where said R⁸ and R^(8a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3substituents selected from the group consisting of F, Cl, Br, I, OH, CN,and N₃; each R⁹ is independently selected from the group consisting ofH, C₁-C₂₀ alkyl,

where each R⁹ C₁-C₂₀ alkyl is optionally substituted by 0 to 3substituents independently selected from the group consisting of OH,—O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; optionallyR^(1a) and R³ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene,C₂-C₆ alkynylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆alkynylene, such that where R^(1a) and R³ are connected to form —O—C₁-C₆alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is boundat the R³ position; optionally R^(2a) and R³ are connected to form C₁-C₆alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene, —O—C₁-C₆ alkylene,—O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R^(2a) andR³ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or—O—C₂-C₆ alkynylene, said O is bound at the R³ position; optionally R³and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene,or —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected toform —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene,said O is bound at the R³ position; optionally R⁴ and R⁵ are connectedto form are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆alkynylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆alkynylene, such that where R⁴ and R⁵ are connected to form —O—C₁-C₆alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is boundat the R⁵ position; optionally R⁵ and R⁶ are connected to form —O—C₁-C₆alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that whereR⁵ and R⁶ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene,or —O—C₂-C₆ alkynylene, said O is bound at the R⁵ position; optionallyR⁷ and R⁸ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, orC₂-C₆ alkynylene; and optionally R^(7a) and R^(8a) are connected to formC₁-C₆ alkylene, C₂-C₆ alkenylene, or C₂-C₆ alkynylene.

In specific aspects of this embodiment, when Y and Y^(a) are each O,X^(a) and X^(a1) are each O, X^(b) and X^(b1) are each O, and X^(c) andX^(c1) are each OH or SH, X^(d) and X^(d1) are each O, R¹ and R^(1a) areeach H, R² is H, R⁶ and R^(6a) are each H, R⁷ and R^(7a) are each H, R⁸and R^(8a) are each H, and Base¹ and Base² are each selected from thegroup consisting of

R⁵ and R³ are not both selected from the group consisting of H, F andOH. That is, when Y and Y^(a) are each O, X^(a) and X^(a1) are each O,X^(b) and X^(b1) are each O, and X^(c) and X^(c1) are each OH or SH,X^(d) and X^(d1) are each O, R¹ and R^(1a) are each H, R² is H, R⁶ andR^(6a) are each H, R⁷ and R^(7a) are each H, R⁸ and R^(8a) are each H,and Base¹ and Base² are each selected from the group consisting of,

either only one of R⁵ and R³ is selected from the group consisting of H,F, and OH, or neither R⁵ and R³ is selected from the group consisting ofH, F, and OH. In further specific instances of this aspect, when Y andY^(a) are each O, X^(a) and X^(a1) are each O, X^(b) and X^(b1) are eachO, and X^(c) and X^(c1) are each OH, X^(d) and X^(d1) are each O or S,R¹ and R^(1a) are each H, R² is H, R⁶ and R^(6a) are each H, R⁷ andR^(7a) are each H, R⁸ and R^(8a) are each H, and Base¹ and Base² areeach selected from the group consisting of

R⁵ and R³ are not both selected from the group consisting of H, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, where said C₁-C₆ alkyl, C₂-C₆alkenyl and C₂-C₆ alkynyl are substituted by 0 to 3 substituentsselected from the group consisting of F, Cl, Br, I and OH.

In further specific aspects of this embodiment, when Base¹ and Base² areeach selected from the group consisting of

and R^(2a) is F and R⁵ is F, at least one of X^(c) and X^(c1) is SR⁹.

In a first aspect of the second embodiment, Base¹ and Base² are eachindependently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3substituents R¹⁰, where each R¹⁰ is independently selected from thegroup consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, andN(C₃₋₆ cycloalkyl)₂. In particular instances, Base¹ and Base² are eachindependently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3substituents R¹⁰, where each R¹⁰ is independently selected from thegroup consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, andN(C₃₋₆ cycloalkyl)₂. In even more particular instances, Base¹ and Base²are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3substituents R¹⁰, where each R¹⁰ is independently selected from thegroup consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, andN(C₃₋₆ cycloalkyl)₂. In this aspect, all other groups are as provided inthe general formula (I′) of the second embodiment above.

In a second aspect of the second embodiment, X^(c) and X^(c1) are eachindependently selected from the group consisting of OR⁹, SR⁹, and NR⁹R⁹,where each R⁹ is independently selected from the group consisting of H,C₁-C₂₀ alkyl,

where each R⁹ C₁-C₂₀ alkyl is optionally substituted by 0 to 3substituents independently selected from the group consisting of OH,—O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl. In particularinstances, X^(c) and X^(c1) are each independently selected from thegroup consisting of O⁻, S⁻,

In all instances of this aspect, all other groups are as provided in thegeneral formula (I′) of the second embodiment above or in the firstaspect described above.

In a third aspect of the second embodiment, R¹ and R^(1a) are each H. Inthis aspect, all other groups are as provided in the general formula(I′) of the second embodiment above or in the first through secondaspects described above.

In a fourth aspect of the second embodiment, R² and R^(2a) are eachindependently selected from the group consisting of H, F, Cl, I, Br, OH,N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R² and R^(2a) C₁-C₆alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selectedfrom the group consisting of F, Cl, Br, I, OH, CN, and N₃. In particularinstances, R² and R^(2a) are each independently selected from the groupconsisting of H, F, Cl, I, Br, OH, CN, N₃, CF₃, CH₃, CH₂OH, and CH₂CH₃.In this aspect, all other groups are as provided in the general formula(I′) of the second embodiment above or in the first through thirdaspects described above.

In a fifth aspect of the second embodiment, R³ is selected from thegroup consisting H, F, Cl, I, Br, OH, N₃, C₁-C₆ alkyl, and C₁-C₆haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substitutedby 0 to 3 substituents selected from the group consisting of F, Cl, Br,I, OH, CN, and N₃. In particular instances, R³ are each independentlyselected from the group consisting of H, F, Cl, I, Br, OH, CN, N₃, CF₃,CH₃, CH₂OH, and CH₂CH₃. In even more particular instances, R³ isselected from NH₂ and N₃. In this aspect, all other groups are asprovided in the general formula (I′) of the second embodiment above orin the first through fourth aspects described above.

In a sixth aspect of the second embodiment, R⁴ and R^(4a) are eachindependently selected from the group consisting of H, F, Cl, I, Br, OH,N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁴ and R^(4a) C₁-C₆alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selectedfrom the group consisting of F, Cl, Br, I, OH, CN, and N₃. In particularinstances, R⁴ and R^(4a) are each independently selected from the groupconsisting of H, F, Cl, I, Br, OH, CN, N₃, CF₃, CH₃, CH₂OH, and CH₂CH₃.In even more particular instances, R⁴ and R^(4a) are each F. In thisaspect, all other groups are as provided in the general formula (I′) ofthe second embodiment above or in the first through fifth aspectsdescribed above.

In a seventh aspect of the second embodiment, R⁵ is selected from thegroup consisting of H, F, Cl, Br, I, OH, NH₂, N₃, C₁-C₆ alkyl, and C₁-C₆haloalkyl, where said R⁵ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substitutedby 0 to 3 substituents selected from the group consisting of F, Cl, Br,I, OH, CN, NR⁹R⁹, and N₃. In particular instances, R⁵ are eachindependently selected from the group consisting of H, F, Cl, I, Br, OH,CN, N₃, CF₃, CH₃, CH₂OH, and CH₂CH₃. In even more particular instances,R⁵ is selected from NH₂ and N₃. In this aspect, all other groups are asprovided in the general formula (I′) of the second embodiment above orin the first through sixth aspects described above.

In an eighth aspect of the second embodiment, R⁶ and R^(6a) are eachindependently selected from the group consisting of H, F, Cl, I, Br, OH,C₁-C₆ alkyl, C₂-C₆ alkenyl, and C₂-C₆ alkynyl. In this aspect, all othergroups are as provided in the general formula (I′) of the secondembodiment above or in the first through seventh aspects describedabove.

In a ninth aspect of the second embodiment, R⁷ and R^(7a) are eachindependently selected from the group consisting of H and C₁-C₆ alkyl.In particular instances, R⁷ and R^(7a) are each independently selectedfrom the group consisting of H and CH₃. In more particular instances,R^(7a) is CH₃. In additional instances, R⁷ and R^(7a) are each H. Inthis aspect, all other groups are as provided in the general formula(I′) of the second embodiment above or in the first through eighthaspects described above.

In a tenth aspect of the second embodiment, R⁸ and R^(8a) are eachindependently selected from the group consisting of H and C₁-C₆ alkyl.In particular instances, R⁸ and R^(8a) are each independently selectedfrom the group consisting of H and CH₃. In more particular instances,R^(8a) is CH₃. In additional instances, R⁸ and R^(8a) are each H. Inthis aspect, all other groups are as provided in the general formula(I′) of the second embodiment above or in the first through ninthaspects described above.

In an eleventh aspect of the second embodiment, R^(1a) and R³ areconnected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene,—O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, suchthat where R^(1a) and R³ are connected to form —O—C₁-C₆ alkylene,—O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³position. In this aspect, all other groups are as provided in thegeneral formula (I′) of the second embodiment above or in the firstthrough tenth aspects described above.

In a twelfth aspect of the second embodiment, R^(2a) and R³ areconnected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene,—O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, suchthat where R^(2a) and R³ are connected to form —O—C₁-C₆ alkylene,—O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³position. In this aspect, all other groups are as provided in thegeneral formula (I′) of the second embodiment above or in the firstthrough eleventh aspects described above.

In a thirteenth aspect of the second embodiment, R³ and R^(6a) areconnected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, and —O—C₂-C₆alkynylene, such that where R³ and R^(6a) are connected to form —O—C₁-C₆alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is boundat the R³ position. In this aspect, all other groups are as provided inthe general formula (I′) of the second embodiment above or in the firstthrough eleventh aspects described above.

In a fourteenth aspect of the second embodiment, R⁴ and R⁵ are connectedby C₁-C₆ alkylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆alkynylene, such that where R⁴ and R⁵ are connected to form —O—C₁-C₆alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is boundat the R⁵ position. In this aspect, all other groups are as provided inthe general formula (I′) of the second embodiment above or in the firstthrough eleventh aspects described above.

In a fifteenth aspect of the second embodiment, R⁵ and R⁶ are connectedto form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene,such that where R⁵ and R⁶ are connected to form —O—C₁-C₆ alkylene,—O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R⁵position. In this aspect, all other groups are as provided in thegeneral formula (I′) of the second embodiment above or in the firstthrough eleventh aspects described above.

In a sixteenth aspect of the second embodiment, R⁷ and R⁸ are connectedto form C₁-C₆ alkylene, C₂-C₆ alkenylene, or C₂-C₆ alkynylene. In thisaspect, all other groups are as provided in the general formula (I′) ofthe second embodiment above or in the first through eleventh aspectsdescribed above.

In a seventeenth aspect of the second embodiment, R^(7a) and R^(8a) areconnected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, or C₂-C₆ alkynylene.In this aspect, all other groups are as provided in the general formula(I′) of the second embodiment above or in the first through eleventhaspects described above.

In an eighteenth aspect of the second embodiment, Base¹ and Base² areeach independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3substituents R¹⁰, where each R¹⁰ is independently selected from thegroup consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, andN(C₃₋₆ cycloalkyl)₂; Y and Y^(a) are each independently selected fromthe group consisting of —O— and —S—; X^(a) and X^(a1) are eachindependently selected from the group consisting of O and S; X^(b) andX^(b1) are each independently selected from the group consisting of Oand S; X^(c) and X^(c1) are each independently selected from the groupconsisting of OR⁹, SR⁹, and NR⁹R⁹; X^(d) and X^(d1) are eachindependently selected from the group consisting of O and S; R¹ andR^(1a) are each H; R² and R^(2a) are each independently selected fromthe group consisting of H, F, Cl, Br, I, OH, N₃, C₁-C₆ alkyl, and C₁-C₆haloalkyl, where said R² and R^(2a) C₁-C₆ alkyl or C₁-C₆ haloalkyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, Br, I, OH, CN, and N₃; R³ is selected from the group consistingof H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, wheresaid R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3substituents selected from the group consisting of F, Cl, Br, I, OH, CN,and N₃; R⁴ and R^(4a) are each independently selected from the groupconsisting of H, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁴ andR^(4a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3substituents selected from the group consisting of F, Cl, Br, I, OH, CN,and N₃; R⁵ is selected from the group consisting of H, F, Cl, Br, I, OH,NH₂, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁵ C₁-C₆ alkyl orC₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from thegroup consisting of F, Cl, Br, I, OH, CN, NR⁹R⁹, and N₃; R⁶ and R^(6a)are each independently selected from the group consisting of H, F, Cl,Br, I, OH, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, and C₁-C₆haloalkyl, where said R⁶ and R^(6a) C₁-C₆ alkyl or C₁-C₆ haloalkyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, Br, I, OH, CN, and N₃; R⁷ and R^(7a) are each H; R⁸ and R^(8a)are each H; each R⁹ is independently selected from the group consistingof H, C₂-C₃ alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2substituents independently selected from the group consisting of OH,—O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; optionally R³and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene,and —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected toform —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene,said O is bound at the R³ position or optionally R⁴ and R⁵ are connectedby C₁-C₆ alkylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆alkynylene, such that where R⁴ and R⁵ are connected to form —O—C₁-C₆alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is boundat the R⁵ position. In all instances of this aspect, all other groupsare as provided in the general formula (I′) of the second embodimentabove.

In a nineteenth aspect of the second embodiment, the compound of formula(I′) is a compound of formula (I′a):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, wherein Base¹ and Base² are each independently selected fromthe group consisting of

where Base¹ and Base² each may be independently substituted by 0-3substituents R¹⁰, where each R¹⁰ is independently selected from thegroup consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, andN(C₃₋₆ cycloalkyl)₂; X^(c) and X^(c1) are each independently selectedfrom the group consisting of OR⁹, SR⁹, and NR⁹R⁹; R³ is selected fromthe group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, andC₁-C₆ haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, Br, I, and OH; R⁵ is selected from the group consisting of H, F,Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where saidR⁵ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituentsselected from the group consisting of F, Cl, Br, I, and OH; R³ and R⁵are not both selected from the group consisting of: OH, R⁵ C₁-C₆ alkylsubstituted with OH, or C₁-C₆ haloalkyl substituted with OH; and each R⁹is independently selected from the group consisting of H, C₂-C₃ alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2substituents independently selected from the group consisting of OH,—O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl. In allinstances of this aspect, all other groups are as provided in thegeneral formula (I′) of the second embodiment above.

In a twentieth aspect of the second embodiment, the compound of formula(I′) is a compound of formula (I′b):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, wherein Base¹ and Base² are each independently selected fromthe group consisting of

where Base¹ and Base² each may be independently substituted by 0-3substituents R¹⁰, where each R¹⁰ is independently selected from thegroup consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, andN(C₃₋₆ cycloalkyl)₂; X^(c) and X^(c1) are each independently selectedfrom the group consisting of OR⁹, SR⁹, and NR⁹R⁹; R^(1a) is selectedfrom the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl,where said R^(1a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3substituents selected from the group consisting of F, Cl, Br, I, OH, CN,and N₃; R^(2a) is selected from the group consisting of H, F, Cl, Br, I,OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R^(2a) C₁-C₆alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selectedfrom the group consisting of F, Cl, Br, I, and OH; R³ is selected fromthe group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, andC₁-C₆ haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, Br, I, and OH; R⁵ is selected from the group consisting of H, F,Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where saidR⁵ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituentsselected from the group consisting of F, Cl, Br, I, and OH; R³ and R⁵are not both selected from the group consisting of OH, C₁-C₆ alkylsubstituted with OH, and C₁-C₆ haloalkyl substituted with OH; R^(6a) isselected from the group consisting of H, F, Cl, Br, I, OH, C₁-C₆ alkyl,C₂-C₆ alkenyl, and C₂-C₆ alkynyl; each R⁹ is independently selected fromthe group consisting of H, C₂-C₃ alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2substituents independently selected from the group consisting of OH,—O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; andoptionally R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene,—O—C₂-C₆ alkenylene, and —O—C₂-C₆ alkynylene, such that where R³ andR^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or—O—C₂-C₆ alkynylene, said O is bound at the R³ position. In this aspect,all other groups are as provided in the general formula (I′) of thesecond embodiment above.

In a twenty-first aspect of the second embodiment, the compound offormula (I′) is a compound of formula (I′c):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, wherein Base¹ and Base² are each independently selected fromthe group consisting of

where Base¹ and Base² each may be independently substituted by 0-3substituents R¹⁰, where each R¹⁰ is independently selected from thegroup consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, andN(C₃₋₆ cycloalkyl)₂; X^(c) and X^(c1) are each independently selectedfrom the group consisting of OR⁹, SR⁹, and NR⁹R⁹; R³ is selected fromthe group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, andC₁-C₆ haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, Br, I, and OH; R⁴ is selected from the group consisting of H, F,Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁴C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituentsselected from the group consisting of F, Cl, Br, I, and OH; R⁵ isselected from the group consisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃,C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁵ C₁-C₆ alkyl or C₁-C₆haloalkyl are substituted by 0 to 3 substituents selected from the groupconsisting of F, Cl, Br, I, and OH; R^(6a) is selected from the groupconsisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆haloalkyl, where said R^(6a) C₁-C₆ alkyl or C₁-C₆ haloalkyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, Br, I, and OH; each R⁹ is independently selected from the groupconsisting of H, C₂-C₃ alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2substituents independently selected from the group consisting of OH,—O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; andoptionally R⁴ and R⁵ are connected by C₁-C₆ alkylene, —O—C₁-C₆ alkylene,—O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R⁴ and R⁵are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or—O—C₂-C₆ alkynylene, said O is bound at the R⁵ position. In this aspect,all other groups are as provided in the general formula (I′) of thesecond embodiment above.

A twenty-second aspect of the second embodiment relates to apharmaceutical composition, said pharmaceutically acceptable compositioncomprising (a) a compound according to any one of general formula (I′)of the second embodiment above or in the first through twenty-firstaspects described above or a pharmaceutically acceptable salt, hydrate,solvate or prodrug thereof, and (b) a pharmaceutically acceptablecarrier.

A twenty-third aspect of the second embodiment relates to methods ofinducing an immune response in a subject, comprising administering atherapeutically effective amount of a compound according to any one ofgeneral formula (I′) of the second embodiment above or in the firstthrough twenty-first aspects described above or a pharmaceuticallyacceptable salt, hydrate, solvate or prodrug thereof to the subject.

A twenty-fourth aspect of the second embodiment relates to methods ofinducing an immune response in a subject, comprising administering atherapeutically effective amount of a composition according to thetwenty-second aspect described above or a pharmaceutically acceptablesalt, hydrate, solvate or prodrug thereof to the subject.

A twenty-fifth aspect of the second embodiment relates to methods ofinducing a STING-dependent type I interferon production in a subject,comprising administering a therapeutically effective amount of acompound according to any one of general (I′) of the second embodimentabove or in the first through twenty-first aspects described above or apharmaceutically acceptable salt, hydrate, solvate or prodrug thereof tothe subject.

A twenty-sixth aspect of the second embodiment relates to methods ofinducing a STING-dependent type I interferon production in a subject,comprising administering a therapeutically effective amount of acomposition according to the twenty-second aspect described above to thesubject.

A twenty-seventh aspect of the second embodiment relates to methods ofinducing a STING-dependent cytokine production in a subject, comprisingadministering a therapeutically effective amount of a compound accordingto any one of general (I′) of the second embodiment above or in thefirst through twenty-first aspects described above or a pharmaceuticallyacceptable salt, hydrate, solvate or prodrug thereof to the subject.

A thirty-third aspect of the second embodiment relates to methods ofinducing a STING-dependent cytokine production in a subject, comprisingadministering a therapeutically effective amount of a compositionaccording to the twenty-second aspect described above to the subject.

A third embodiment of the disclosure relates to cyclic di-nucleotidecompounds of general formula (I″):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, wherein Base¹ and Base² are each independently selected fromthe group consisting of

Y is selected from the group consisting of —O— and —S—; X^(c) and X^(c1)are each independently selected from the group consisting of OR⁹ andSR⁹; X^(d) and X^(d1) are each independently selected from the groupconsisting of O and S; R^(2a) is selected from the group consisting ofH, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R³ is selected from the groupconsisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl,—O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R⁴ is selectedfrom the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R⁵is selected from the group consisting of H, F, Cl, Br, I, OH, CN, NH₂,N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and—O—C₂-C₆ alkynyl; R^(6a) is selected from the group consisting of H, F,Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; each R⁹ is independentlyselected from the group consisting of H, C₁-C₂₀ alkyl,

where each R⁹ C₁-C₂₀ alkyl is optionally substituted by 0 to 3substituents independently selected from the group consisting of OH,—O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; andoptionally R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene,—O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R³ andR^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or—O—C₂-C₆ alkynylene, said O is bound at the R³ position.

In specific aspects of this embodiment, when Y and Y^(a) are each O,X^(a) and X^(a1) are each O, X^(b) and X^(b1) are each O, and X^(c) andX^(c1) are each OH or SH, X^(d) and X^(d1) are each O, R¹ and R^(1a) areeach H, R² is H, R⁶ and R^(6a) are each H, R⁷ and R^(7a) are each H, R⁸and R^(8a) are each H, and Base¹ and Base² are each selected from thegroup consisting of

R⁵ and R³ are not both selected from the group consisting of H, F andOH. That is, when Y and Y^(a) are each O, X^(a) and X^(a1) are each O,X^(b) and X^(b1) are each O, and X^(c) and X^(c1) are each OH or SH,X^(d) and X^(d1) are each O, R¹ and R^(1a) are each H, R² is H, R⁶ andR^(6a) are each H, R⁷ and R^(7a) are each H, R⁸ and R^(8a) are each H,and Base¹ and Base² are each selected from the group consisting of

either only one of R⁵ and R³ is selected from the group consisting of H,F, and OH, or neither R⁵ and R³ is selected from the group consisting ofH, F, and OH. In further specific instances of this aspect, when Y andY^(a) are each O, X^(a) and X^(a1) are each O, X^(b) and X^(b1) are eachO, and X^(c) and X^(c1) are each OH, X^(d) and X^(d1) are each O or S,R¹ and R^(1a) are each H, R² is H, R⁶ and R^(6a) are each H, R⁷ andR^(7a) are each H, R⁸ and R^(8a) are each H, and Base¹ and Base² areeach selected from the group consisting of

R⁵ and R³ are not both selected from the group consisting of H, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, where said C₁-C₆ alkyl, C₂-C₆alkenyl and C₂-C₆ alkynyl are substituted by 0 to 3 substituentsselected from the group consisting of F, Cl, Br, I and OH.

In further specific aspects of this embodiment, when Base¹ and Base² areeach selected from the group consisting of

and R^(2a) is F and R⁵ is F, at least one of X^(c) and X^(c1) is SR⁹.

In a first aspect of the third embodiment, the compound of formula (I″)is a compound of formula (I″a):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, wherein Base¹ and Base² are each independently selected fromthe group consisting of

Y is selected from the group consisting of —O— and —S—; X^(c) and X^(c1)are each independently selected from the group consisting of OR⁹ andSR⁹; X^(d) and X^(d1) are each independently selected from the groupconsisting of O and S; R^(2a) is selected from the group consisting ofH, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R⁵ is selected from the groupconsisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl;R^(6a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN,N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and—O—C₂-C₆ alkynyl; and each R⁹ is independently selected from the groupconsisting of H, C₁-C₂₀ alkyl,

where each R⁹ C₁-C₂₀ alkyl is optionally substituted by 0 to 3substituents independently selected from the group consisting of OH,—O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl. In instancesof this aspect, Base¹ and Base² are each independently selected from thegroup consisting of

Y is selected from the group consisting of —O— and —S—; X^(c) and X^(c1)are each independently selected from the group consisting of OR⁹ andSR⁹; X^(d) and X^(d1) are each independently selected from the groupconsisting of O and S; R^(2a) is F; R⁵ is selected from the groupconsisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl;R^(6a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN,N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and—O—C₂-C₆ alkynyl; and each R⁹ is independently H.

In a second aspect of the third embodiment, the compound of formula (I″)is a compound wherein R³ and R^(6a) are connected to form —O—C₁-C₆alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that whereR³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³ position.

In a third aspect of the third embodiment, the compound of formula (I″)is a compound of formula (I″b):

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, wherein Base¹ and Base² are each independently selected fromthe group consisting of

Y is selected from the group consisting of —O— and —S—; X^(c) and X^(c1)are each independently selected from the group consisting of OR⁹ andSR⁹; X^(d) and X^(d1) are each independently selected from the groupconsisting of O and S; R³ is selected from the group consisting of H, F,Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R⁴ is selected from the groupconsisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl,—O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R⁵ is selectedfrom the group consisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl,C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆alkynyl; R^(6a) is selected from the group consisting of H, F, Cl, Br,I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; each R⁹ is independently H; andR³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆alkenylene, or —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) areconnected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆alkynylene, said O is bound at the R³ position. In instances of thisaspect, Base¹ and Base² are each independently selected from the groupconsisting of

In a fourth aspect of the third embodiment, the compound of formula (I″)is a compound wherein at least one of Base¹ and Base² are eachindependently selected from the group consisting of

A fifth aspect of the third embodiment relates to a pharmaceuticalcomposition, said pharmaceutical composition comprising (a) a compoundaccording to any one of general formula (I″) of the third embodiment ora pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof;and (b) a pharmaceutically acceptable carrier.

A sixth aspect of the third embodiment relates to methods of inducing animmune response in a subject, comprising administering a therapeuticallyeffective amount of a compound according to general formula (I″) of thethird embodiment above or a pharmaceutically acceptable salt, hydrate,solvate, or prodrug thereof to the subject.

A seventh aspect of the third embodiment relates to methods of inducingan immune response in a subject, comprising administering atherapeutically effective amount of a composition according to the fifthaspect described above to the subject.

An eighth aspect of the third embodiment relates to methods of inducinga STING-dependent type I interferon production in a subject, comprisingadministering a therapeutically effective amount of a compound accordinggeneral formula (I″) of the third embodiment above or a pharmaceuticallyacceptable salt, hydrate, solvate, or prodrug thereof to the subject.

A ninth aspect of the third embodiment relates to methods of inducing aSTING-dependent type I interferon production in a subject, comprisingadministering a therapeutically effective amount of a compositionaccording to the fifth aspect described above to the subject.

A tenth aspect of the third embodiment relates to methods of inducing aSTING-dependent cytokine production in a subject, comprisingadministering a therapeutically effective amount of a compound accordingto general formula (I″) of the third embodiment above or apharmaceutically acceptable salt, hydrate, solvate, or prodrug thereofto the subject.

An eleventh aspect of the third embodiment relates to methods ofinducing a STING-dependent cytokine production in a subject, comprisingadministering a therapeutically effective amount of a compositionaccording to the fifth aspect described above to the subject.

In an additional embodiment, the compound is selected from the groupconsisting of

and pharmaceutically acceptable salts, hydrates, solvates, or prodrugsthereof. In aspects of this embodiment, the compound is selected fromthe group consisting of

and pharmaceutically acceptable salts, hydrates, solvates, or prodrugsthereof. In particular aspects, the compound is selected from the groupconsisting of

and pharmaceutically acceptable salts, hydrates, solvates, or prodrugsthereof. In more particular aspects of this embodiment, the compound isselected from the group consisting of

and pharmaceutically acceptable salts, hydrates, solvates, or prodrugsthereof. In still more particular aspects, the compound is selected fromthe group consisting of

and pharmaceutically acceptable salts, hydrates, solvates, or prodrugsthereof.

In another embodiment, for the compounds of general formula (I),compounds of general formula (I′) and compounds of general formula (I″),variables Base¹, Base², Y, Y^(a), X^(a), X^(a1), X^(b), X^(b1), X^(c),X^(c1), X^(d), X^(d1), R¹, R^(1a), R², R^(2a), R³, R⁴, R^(4a), R⁵, R⁶,R^(6a), R⁷, R^(7a), R⁸, R^(8a), R⁹, and R¹⁰ are each selectedindependently from each other.

In another embodiment of the disclosure, the compound of the disclosureis selected from the exemplary species depicted in Examples 1 through348 shown below.

Other embodiments of the present disclosure include the following:

(a) A pharmaceutical composition comprising an effective amount of acompound of general formula (I) or a compound of general formula (I′),or a compound of general formula (I″), or a pharmaceutically acceptablesalt, hydrate, solvate, or prodrug thereof, and a pharmaceuticallyacceptable carrier.

(b) The pharmaceutical composition of (a), further comprising a secondtherapeutic agent selected from the group consisting of STING agonistcompounds, anti-viral compounds, antigens, adjuvants, CTLA-4 and PD-1pathway antagonists and other immunomodulatory agents, lipids,liposomes, peptides, anti-cancer and chemotherapeutic agents.

(c) A pharmaceutical combination that is (i) a compound of generalformula (I) or a compound of general formula (I′), or a compound ofgeneral formula (I″), or a pharmaceutically acceptable salt, hydrate,solvate, or prodrug thereof, and (ii) a second therapeutic agentselected from the group consisting of STING agonist compounds,anti-viral compounds, antigens, adjuvants, CTLA-4 and PD-1 pathwayantagonists and other immunomodulatory agents, lipids, liposomes,peptides, anti-cancer and chemotherapeutic agents; wherein the acompound of general formula (I) or compound of general formula (I′), orcompound of general formula (I″), or a pharmaceutically acceptable salt,hydrate, solvate, or prodrug thereof, and the second therapeutic agentare each employed in an amount that renders the combination effectivefor inducing an immune response in a patient.

(e) A method of inducing an immune response in a patient, whichcomprises administering to the subject an effective amount of a compoundof general formula (I) or a compound of general formula (I′), or acompound of general formula (I″), or a pharmaceutically acceptable salt,hydrate, solvate, or prodrug thereof.

(f) A method of inducing an immune response in a patient, whichcomprises administering to the subject an effective amount of acomposition of (a), a composition of (b) or a combination of (c).

(g) A method of inducing STING-dependent type I interferon production ina patient, which comprises administering to the subject an effectiveamount of (a), a compound of general formula (I) or a compound ofgeneral formula (I′), or a compound of general formula (I″).

(h) A method of inducing STING-dependent type I interferon production ina patient, which comprises administering to the subject an effectiveamount of a composition of (a), a composition of (b) or a combination of(c).

(i) A method of inducing STING-dependent cytokine production in apatient, which comprises administering to the subject an effectiveamount of a compound of general formula (I) or a compound of generalformula (I′), or a compound of general formula (I″), or apharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.

(j) A method of inducing STING-dependent cytokine production in apatient, which comprises administering to the subject an effectiveamount of a composition of (a), a composition of (b) or a combination of(c).

(k) A method of treating a cell proliferation disorder in a subject,said method comprising administering a therapeutically effective amountof a compound of general formula (I) or a compound of general formula(I′), or a compound of general formula (I″), or a pharmaceuticallyacceptable salt, hydrate, solvate, or prodrug thereof to the subject;

(l) The method of (k), wherein the cell proliferation disorder iscancer.

(m). A method of treating a cell proliferation disorder in a subject,said method comprising administering a therapeutically effective amountof a pharmaceutical composition of (a), a composition of (b) or acombination of (c) to the subject.

(n) The method of (m), wherein the cell proliferation disorder iscancer.

The present disclosure also includes a compound of the presentdisclosure for use (i) in, (ii) as a medicament for, or (iii) in thepreparation of a medicament for: (a) inducing an immune response in apatient, or (b) inducing a STING-dependent cytokine production in apatient. In these uses, the compounds of the present disclosure canoptionally be employed in combination with one or more secondtherapeutic agents selected from STING agonist compounds, anti-viralcompounds, antigens, adjuvants, CTLA-4 and PD-1 pathway antagonists andother immunomodulatory agents, lipids, liposomes, peptides, anti-cancerand chemotherapeutic agents.

Additional embodiments of the disclosure include the pharmaceuticalcompositions, combinations and methods set forth in (a)-(n) above andthe uses set forth in the preceding paragraph, wherein the compound ofthe present disclosure employed therein is a compound of one of theembodiments, aspects, classes, sub-classes, or features of the compoundsdescribed above. In all of these embodiments, the compound mayoptionally be used in the form of a pharmaceutically acceptable salt,hydrate, solvate or prodrug as appropriate.

In the embodiments of the compound provided above, it is to beunderstood that each embodiment may be combined with one or more otherembodiments, to the extent that such a combination provides a stablecompound and is consistent with the description of the embodiments. Itis further to be understood that the embodiments of compositions andmethods provided as (a) through (n) above are understood to include allembodiments of the compounds, including such embodiments as result fromcombinations of embodiments.

The term “subject” (alternatively referred to herein as “patient”) asused herein refers to an animal, preferably a mammal, such as a humanbeing, male or female, that has been the object of treatment,observation, or experiment. A subject also refers to one or more ofcows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, andbirds. In embodiments, the subject is human.

As used herein, the term “immune response” relates to any one or more ofthe following: specific immune response, non-specific immune response,both specific and non-specific response, innate response, primary immuneresponse, adaptive immunity, secondary immune response, memory immuneresponse, immune cell activation, immune cell proliferation, immune celldifferentiation, and cytokine expression. In certain embodiments, thecompound of general formula (I), compound of general formula (I′), orcompound of general formula (I″), or pharmaceutically acceptable salt,hydrate, solvate, or prodrug thereof, is administered in conjunctionwith one or more additional therapeutic agents including vaccinesintended to stimulate an immune response to one or more predeterminedanti-viral compounds, antigens, adjuvants, CTLA-4 and PD-1 pathwayantagonists and other immunomodulatory agents, lipids, liposomes,peptides, anti-cancer and chemotherapeutic agents, etc. In certainembodiments, the compound of general formula (I), compound of generalformula (I′), or compound of general formula (I″), or a pharmaceuticallyacceptable salt, hydrate, solvate, or prodrug thereof, is administeredin conjunction with one or more additional compositions includingvaccines intended to stimulate an immune response to one or morepredetermined anti-viral compounds, antigens, adjuvants, CTLA-4 and PD-1pathway antagonists and other immunomodulatory agents, lipids,liposomes, peptides, anti-cancer and chemotherapeutic agents, etc.

Compounds

The term “alkyl” refers to a monovalent straight or branched chain,saturated aliphatic hydrocarbon radical having a number of carbon atomsin the specified range. Thus, for example, “C₁₋₆ alkyl” (or “C₁-C₆alkyl”) refers to any of the hexyl alkyl and pentyl alkyl isomers aswell as n-, iso-, sec- and tert-butyl, n- and iso-propyl, ethyl, andmethyl. As another example, “C₁₋₄ alkyl” refers to n-, iso-, sec- andtert-butyl, n- and isopropyl, ethyl, and methyl.

As used herein, the term “alkylene” refers to a bivalent straight chain,saturated aliphatic hydrocarbon radical having a number of carbon atomsin the specified range.

As used herein, the term “alkenyl” refers to a monovalent straight orbranched chain, unsaturated aliphatic hydrocarbon radical having anumber of carbon atoms in the specified range and including one or moredouble bond.

As used herein, the term “alkenylene” refers to a bivalent straightchain, unsaturated aliphatic hydrocarbon radical having a number ofcarbon atoms in the specified range and including one or more doublebond.

As used herein, the term “alkynyl” refers to a monovalent straight orbranched chain, unsaturated aliphatic hydrocarbon radical having anumber of carbon atoms in the specified range and including one or moretriple bond.

As used herein, the term “alkynylene” refers to a bivalent straightchain, unsaturated aliphatic hydrocarbon radical having a number ofcarbon atoms in the specified range and including one or more triplebond.

The term “halogen” (or “halo”) refers to fluorine, chlorine, bromine,and iodine (alternatively referred to as fluoro, chloro, bromo, and iodoor F, Cl, Br, and I).

The term “haloalkyl” refers to an alkyl group as defined above in whichone or more of the hydrogen atoms have been replaced with a halogen.Thus, for example, “C₁₋₆ haloalkyl” (or “C₁-C₆ haloalkyl”) refers to aC₁ to C₆ linear or branched alkyl group as defined above with one ormore halogen substituents. The term “fluoroalkyl” has an analogousmeaning except the halogen substituents are restricted to fluoro.Suitable fluoroalkyls include the series (CH₂)₀₋₄CF₃ (i.e.,trifluoromethyl, 2,2,2-trifluoroethyl, 3,3,3-trifluoro-n-propyl, etc.).

As used herein, the term “haloalkenyl” refers to an alkenyl group asdefined above in which one or more of the hydrogen atoms have beenreplaced with a halogen.

As used herein, the term “haloalkynyl” refers to an alkynyl group asdefined above in which one or more of the hydrogen atoms have beenreplaced with a halogen.

As used herein, the term “spirocycle” or “spirocyclic ring” refers to apendant cyclic group formed by substituents on a single atom. Forexample, in general formula (I), a spirocycle may be formed by R^(2a)and R³.

Unless expressly stated to the contrary, all ranges cited herein areinclusive; i.e., the range includes the values for the upper and lowerlimits of the range as well as all values in between. As an example,temperature ranges, percentages, ranges of equivalents, and the likedescribed herein include the upper and lower limits of the range and anyvalue in the continuum there between. Numerical values provided herein,and the use of the term “about”, may include variations of ±1%, ±2%,±3%, ±4%, ±5%, ±10%, ±15%, and ±20% and their numerical equivalents.

As used herein, the term “one or more” item includes a single itemselected from the list as well as mixtures of two or more items selectedfrom the list.

In the compounds of general formula (I), compounds of general formula(I′), and compounds of general formula (I″), the atoms may exhibit theirnatural isotopic abundances, or one or more of the atoms may beartificially enriched in a particular isotope having the same atomicnumber, but an atomic mass or mass number different from the atomic massor mass number predominantly found in nature. The present disclosure ismeant to include all suitable isotopic variations of the compounds ofgeneral formula (I), compounds of general formula (I′), and compounds ofgeneral formula (I″). For example, different isotopic forms of hydrogen(H) include protium (¹H) and deuterium (²H). Protium is the predominanthydrogen isotope found in nature. Enriching for deuterium may affordcertain therapeutic advantages, such as increasing in vivo half-life orreducing dosage requirements, or may provide a compound useful as astandard for characterization of biological samples.Isotopically-enriched compounds within general formula (I) can beprepared without undue experimentation by conventional techniques wellknown to those skilled in the art or by processes analogous to thosedescribed in the Schemes and Examples herein using appropriateisotopically-enriched reagents and/or intermediates.

In particular embodiments of the compounds of general formula (I),compounds of general formula (I′), and compounds of general formula(I″), the compounds are isotopically enriched with deuterium. In aspectsof these embodiments, one or more of R¹, R^(1a), R², R^(2a), R³, R⁴,R^(4a), R⁵, R⁶, R^(6a), R⁷, R^(7a), R⁸, R^(8a), R⁹, and R¹⁰ may bedeuterium.

As shown in the general structural formulas and the structures ofspecific compounds as provided herein, a straight line at a chiralcenter includes both (R) and (S) stereoisomers and mixtures thereof.Also, unless otherwise specified (e.g., 100% purified compound),reference to a particular stereochemistry at a position provides acompound having the indicated stereochemistry, but does not exclude thepresence of stereoisomers having different stereochemistry at theindicated position.

Recitation or depiction of a specific compound in the claims (i.e., aspecies) without a specific stereoconfiguration designation, or withsuch a designation for less than all chiral centers, is intended toencompass the racemate, racemic mixtures, each individual enantiomer, adiastereoisomeric mixture and each individual diastereomer of thecompound where such forms are possible due to the presence of one ormore asymmetric centers. The separation of a mixture of stereoisomerscan be carried out at an intermediate step during the synthesis of acompound of general formula (I), a compound of general formula (I′),and/or a compound of general formula (I″), or it can be done on a finalracemic product. Absolute stereochemistry may be determined by X-raycrystallography of crystalline products or crystalline intermediates,which are derivatized, if necessary, with a reagent containing astereogenic center of known configuration. Alternatively, absolutestereochemistry may be determined by Vibrational Circular Dichroism(VCD) spectroscopy analysis. The present invention includes all suchisomers, as well as salts, solvates (which includes hydrates) andsolvated salts of such racemates, enantiomers, diastereomers andtautomers and mixtures thereof.

Those skilled in the art will recognize that chiral compounds, and inparticular sugars, can be drawn in a number of different ways that areequivalent. Those skilled in the art will further recognize that theidentity and regiochemical position of the substituents on ribose canvary widely and that the same principles of stereochemical equivalenceapply regardless of substituent. Non-limiting examples of suchequivalence include those exemplified below.

Salts

Compounds described herein having appropriate functional groups can beprovided as salts. Examples of such compounds are described herein byreference to possible salts. Such reference is for illustration only.Additional embodiments include salts of any compounds described hereinhaving suitable groups.

Pharmaceutically acceptable salts can be used with compounds fortreating patients. Non-pharmaceutical salts may, however, be useful inthe preparation of intermediate compounds.

Pharmaceutically acceptable salts are suitable for administration to apatient, preferably, a human. Suitable salts include acid addition saltsthat may, for example, be formed by mixing a solution of a compound witha solution of a pharmaceutically acceptable acid such as hydrochloricacid, sulfuric acid, acetic acid, trifluoroacetic acid, or benzoic acid.Compounds carrying an acidic moiety can be mixed with suitablepharmaceutically acceptable salts to provide, for example, alkali metalsalts (e.g., sodium or potassium salts), alkaline earth metal salts(e.g., calcium or magnesium salts), and salts formed with suitableorganic ligands such as quaternary ammonium salts. Also, in the case ofan acid (—COOH) or alcohol group being present, pharmaceuticallyacceptable esters can be employed to modify the solubility or hydrolysischaracteristics of the compound.

Methods of Preparing Compounds

Several methods for preparing the compounds of general formula (I),compounds of general formula (I′), and compounds of general formula(I″), or pharmaceutically acceptable salts, hydrates, solvates, orprodrugs thereof, are described in the following Schemes and Examples.Starting materials and intermediates are purchased from commercialsources, made from known procedures, or are otherwise illustrated. Insome cases the order of carrying out the steps of the reaction schemesmay be varied to facilitate the reaction or to avoid unwanted reactionproducts.

Method 1

One method for the preparation of examples of the disclosure is detailedin Scheme 1. This procedure was adequately modified from the previouslyreported procedure for cyclic dinucleotide synthesis (Barbara L. Gaffneyet al., One-Flask Syntheses of c-di-GMP and the [Rp,Rp] and [Rp,Sp]Thiophosphate Analogues, 12 ORG. LETT. 3269-3271 (2010)). The sequencestarts with modified ribo-nucleoside with a nucleobase of which aminogroup was appropriately protected with an alkyl or phenyl carbonylgroup, a phosphoramidite functionality at 2′-O position, and DMTr etherat 5′-O position. It was treated with aqueous TFA/pyridine condition andsubsequently t-butylamine to convert the 2′-phosphoramidite moiety to anH-phosphonate. Then, DMTr ether was removed under acidic condition. Theresulting 5′-hydroxyl group was reacted with 3′-phosphoramidites offully protected second modified ribo-nucleoside to give a cyclizedcompound. It was immediately oxidized with t-butyl hydroperoxide. Then,the 5′-hydroxyl group of the second ribo-nucleoside was deprotected withdichloroacetic acid. Using 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane2-oxide as a coupling reagent, the H-phosphonate at 2′-O of the firstribo-nucleoside was reacted with 5′-OH of the second ribo-nucleoside togive a cyclic product. It was immediately oxidized with aqueous iodine.Treatment with t-butylamine and methylamine plus fluoride anion in casesilyl protection was used provided the desired cyclic dinucleotide 1G.

Method 2

Another method for the preparation of examples of the disclosure isdetailed in Scheme 2. This procedure was modified from Scheme 1. Thesequence starts with modified ribo-nucleoside with a nucleobase of whichamino group was appropriately protected with an alkyl or phenyl carbonylgroup, a phosphoramidite functionality at 2′-O position, and DMTr etherat 5′-O position. It was treated with aqueous TFA/pyridine condition andsubsequently t-butylamine to convert the 2′-phosphoramidite moiety to anH-phosphonate. Then, DMTr ether was removed under acidic condition. Theresulting 5′-hydroxyl group was reacted with 3′-phosphoramidites offully protected second modified ribo-nucleoside to give a cyclizedcompound. It was immediately thioated with(E)-N,N-dimethyl-N′-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide.Then, the 5′-hydroxyl group of the second ribo-nucleoside wasdeprotected with dichloroacetic acid. Using2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide as a couplingreagent, the H-phosphonate at 2′-O of the first ribo-nucleoside wasreacted with 5′-OH of the second ribo-nucleoside to give a cyclicproduct. It was immediately thioated with 3H-benzo[c][1,2]dithiol-3-one.Treatment with t-butylamine and methylamine plus fluoride anion in casesilyl protection was used provided the desired cyclic dinucleotidediphosphorothioate 2G.

Methods of Use

Compounds described herein having therapeutic applications, such as thecompounds of general formula (I), compounds of general formula (I′), andcompounds of general formula (I″), and the compounds of the Examples 1through 348, can be administered to a patient for the purpose ofinducing an immune response, inducing a STING-dependent cytokineproduction and/or inducing anti-tumor activity. The term“administration” and variants thereof (e.g., “administering” a compound)means providing the compound to the individual in need of treatment.When a compound is provided in combination with one or more other activeagents (e.g., antiviral agents useful for treating HCV infection oranti-tumor agents for treating cancers), “administration” and itsvariants are each understood to include concurrent and sequentialprovision of the compound or salt and other agents.

The compounds disclosed herein are STING agonists and inhibitors ofviral replication. These compounds are potentially useful in treatingdiseases or disorders including, but not limited to, cell proliferationdisorders, such as cancer.

Cell-proliferation disorders include, but are not limited to, cancer.Examples of such cancers include, but are not limited to, AcuteLymphoblastic Leukemia; Acute Myeloid Leukemia; AdrenocorticalCarcinoma; AIDS-Related Lymphoma; AIDS-Related Malignancies; AnalCancer; Astrocytoma; Bile Duct Cancer; Bladder Cancer; Bone Cancer,Osteosarcoma/Malignant Fibrous Histiocytoma; Brain Stem Glioma; BrainTumor, Cerebellar Astrocytoma; Brain Tumor, CerebralAstrocytoma/Malignant Glioma; Brain Tumor, Ependymoma; Brain Tumor,Medulloblastoma; Brain Tumor, Supratentorial Primitive NeuroectodermalTumors; Brain Tumor, Visual Pathway and Hypothalamic Glioma; BreastCancer; Bronchial Adenomas/Carcinoids; Carcinoid Tumor; Carcinoid Tumor,Gastrointestinal; Carcinoma, Adrenocortical; Carcinoma, Islet Cell;Central Nervous System Lymphoma, Primary; Cerebral Astrocytoma/MalignantGlioma; Cervical Cancer; Chronic Lymphocytic Leukemia; ChronicMyelogenous Leukemia; Chronic Myeloproliferative Disorders; Clear CellSarcoma of Tendon Sheaths; Colon Cancer; Colorectal Cancer; CutaneousT-Cell Lymphoma; Endometrial Cancer; Ependymoma; Epithelial Cancer,Ovarian; Esophageal Cancer; Esophageal Cancer; Ewing's Family of Tumors;Extracranial Germ Cell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer,Intraocular Melanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer;Gastric (Stomach) Cancer; Gastrointestinal Carcinoid Tumor; Germ CellTumor, Extracranial, Childhood; Germ Cell Tumor, Extragonadal; Germ CellTumor, Ovarian; Gestational Trophoblastic Tumor; Glioma, Childhood BrainStem; Glioma, Childhood Visual Pathway and Hypothalamic; Hairy CellLeukemia; Head and Neck Cancer; Hepatocellular (Liver) Cancer; Hodgkin'sLymphoma; Hypopharyngeal Cancer; Hypothalamic and Visual Pathway Glioma;Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas);Kaposi's Sarcoma; Kidney Cancer; Laryngeal Cancer; Leukemia, AcuteLymphoblastic; Leukemia, Acute Myeloid; Leukemia, Chronic Lymphocytic;Leukemia, Chronic Myelogenous; Leukemia, Hairy Cell; Lip and Oral CavityCancer; Liver Cancer; Lung Cancer, Non-Small Cell; Lung Cancer, SmallCell; Lymphoblastic Leukemia; Lymphoma, AIDS-Related; Lymphoma, CentralNervous System (Primary); Lymphoma, Cutaneous T-Cell; Lymphoma,Hodgkin's; Lymphoma, Hodgkin's During Pregnancy; Lymphoma,Non-Hodgkin's; Lymphoma, Primary Central Nervous System;Macroglobulinemia, Waldenstrom's; Male Breast Cancer; MalignantMesothelioma; Malignant Thymoma; Medulloblastoma, Childhood; Melanoma;Melanoma, Intraocular; Merkel Cell Carcinoma; Mesothelioma, Malignant;Metastatic Squamous Neck Cancer with Occult Primary; Multiple EndocrineNeoplasia Syndrome, Childhood; Multiple Myeloma/Plasma Cell Neoplasm;Mycosis Fungoides; Myelodysplastic Syndromes; Myelogenous Leukemia,Chronic; Myeloid Leukemia; Myeloma, Multiple; MyeloproliferativeDisorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer;Nasopharyngeal Cancer; Neuroblastoma; Non-Hodgkin's Lymphoma; Non-SmallCell Lung Cancer; Oral Cancer; Oral Cavity and Lip Cancer; OropharyngealCancer; steosarcoma/Malignant Fibrous Histiocytoma of Bone; OvarianEpithelial Cancer; Ovarian Germ Cell Tumor; Ovarian Low MalignantPotential Tumor; Pancreatic Cancer; Paranasal Sinus and Nasal CavityCancer; Parathyroid Cancer; Penile Cancer; Pheochromocytoma; Pineal andSupratentorial Primitive Neuroectodermal Tumors; Pituitary Tumor; PlasmaCell Neoplasm/Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy andBreast Cancer; Pregnancy and Hodgkin's Lymphoma; Pregnancy andNon-Hodgkin's Lymphoma; Primary Central Nervous System Lymphoma; PrimaryLiver Cancer; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney)Cancer; Renal Pelvis and Ureter, Transitional Cell Cancer;Retinoblastoma; Rhabdomyosarcoma; Salivary Gland Cancer; Sarcoma,Ewing's Family of Tumors; Sarcoma, Kaposi's; Sarcoma(Osteosarcoma)/Malignant Fibrous Histiocytoma of Bone; Sarcoma, SoftTissue; Sezary Syndrome; Skin Cancer; Skin Cancer (Melanoma); SkinCarcinoma, Merkel Cell; Small Cell Lung Cancer; Small Intestine Cancer;Soft Tissue Sarcoma; Squamous Neck Cancer with Occult Primary,Metastatic; Stomach (Gastric) Cancer; Supratentorial PrimitiveNeuroectodermal Tumors; T-Cell Lymphoma, Cutaneous; Testicular Cancer;Thymoma, Malignant; Thyroid Cancer; Transitional Cell Cancer of theRenal Pelvis and Ureter; Trophoblastic Tumor, Gestational; Ureter andRenal Pelvis, Transitional Cell Cancer; Urethral Cancer; UterineSarcoma; Vaginal Cancer; Visual Pathway and Hypothalamic Glioma; VulvarCancer; Waldenstrom's Macro globulinemia; and Wilms' Tumor.

In one embodiment, the cancer is brain cancer, such as an astrocytictumor (e.g., pilocytic astrocytoma, subependymal giant-cell astrocytoma,diffuse astrocytoma, pleomorphic xanthoastrocytoma, anaplasticastrocytoma, astrocytoma, giant cell glioblastoma, glioblastoma,secondary glioblastoma, primary adult glioblastoma, and primarypediatric glioblastoma); oligodendroglial tumor (e.g.,oligodendroglioma, and anaplastic oligodendroglioma); oligoastrocytictumor (e.g., oligoastrocytoma, and anaplastic oligoastrocytoma);ependymoma (e.g., myxopapillary ependymoma, and anaplastic ependymoma);medulloblastoma; primitive neuroectodermal tumor, schwannoma,meningioma, atypical meningioma, anaplastic meningioma; and pituitaryadenoma. In another embodiment, the brain cancer is glioma, glioblastomamultiforme, paraganglioma, or suprantentorial primordial neuroectodermaltumors (sPNET).

In another embodiment, the cancer is leukemia, such as acute myeloidleukemia (AML), myelodysplastic syndrome (MDS), chronic myelogenousleukemia (CML), myeloproliferative neoplasm (MPN), post-MPN AML,post-MDS AML, del(5q)-associated high risk MDS or AML, blast-phasechronic myelogenous leukemia, angioimmunoblastic lymphoma, and acutelymphoblastic leukemia.

In one embodiment, the cancer is skin cancer, including melanoma. Inanother embodiment, the cancer is prostate cancer, breast cancer,thyroid cancer, colon cancer, or lung cancer. In another embodiment, thecancer is sarcoma, including central chondrosarcoma, central andperiosteal chondroma, and fibrosarcoma. In another embodiment, thecancer is cholangiocarcinoma.

As used herein, the terms “treatment” and “treating” refer to allprocesses wherein there may be a slowing, interrupting, arresting,controlling, or stopping of the progression of a disease or disorderdescribed herein. The terms do not necessarily indicate a totalelimination of all disease or disorder symptoms.

The terms “administration of” and or “administering” a compound shouldbe understood to include providing a compound described herein, or apharmaceutically acceptable salt, hydrate, solvate or prodrug thereof,and compositions of the foregoing to a subject.

The amount of a compound administered to a subject is an amountsufficient to induce an immune response and/or to induce STING-dependenttype I interferon production in the subject. In an embodiment, theamount of a compound can be an “effective amount” or “therapeuticallyeffective amount,” wherein the subject compound is administered in anamount that will elicit, respectively, a biological or medical (i.e.,intended to treat) response of a tissue, system, animal or human that isbeing sought by a researcher, veterinarian, medical doctor or otherclinician. An effective amount does not necessarily includeconsiderations of toxicity and safety related to the administration of acompound.

An effective amount of a compound will vary with the particular compoundchosen (e.g., considering the potency, efficacy, and/or half-life of thecompound); the route of administration chosen; the condition beingtreated; the severity of the condition being treated; the age, size,weight, and physical condition of the subject being treated; the medicalhistory of the subject being treated; the duration of the treatment; thenature of a concurrent therapy; the desired therapeutic effect; and likefactors and can be routinely determined by the skilled artisan.

The compounds disclosed herein may be administered by any suitable routeincluding oral and parenteral administration. Parenteral administrationis typically by injection or infusion and includes intravenous,intramuscular, and subcutaneous injection or infusion.

The compounds disclosed herein may be administered once or according toa dosing regimen wherein a number of doses are administered at varyingintervals of time for a given period of time. For example, doses may beadministered one, two, three, or four times per day. Doses may beadministered until the desired therapeutic effect is achieved orindefinitely to maintain the desired therapeutic effect. Suitable dosingregimens for a compound disclosed herein depend on the pharmacokineticproperties of that compound, such as absorption, distribution andhalf-life which can be determined by a skilled artisan. In addition,suitable dosing regimens, including the duration such regimens areadministered, for a compound disclosed herein depend on the disease orcondition being treated, the severity of the disease or condition, theage and physical condition of the subject being treated, the medicalhistory of the subject being treated, the nature of concurrent therapy,the desired therapeutic effect, and like factors within the knowledgeand expertise of the skilled artisan. It will be further understood bysuch skilled artisans that suitable dosing regimens may requireadjustment given an individual subject's response to the dosing regimenor over time as the individual subject needs change. Typical dailydosages may vary depending upon the particular route of administrationchosen.

One embodiment of the present disclosure provides for a method oftreating a cell proliferation disorder comprising administration of atherapeutically effective amount of a compound of general formula (I), acompound of general formula (I′), or a compound of general formula (I″)to a subject in need of treatment thereof. In one embodiment, the cellproliferation disorder is cancer.

In one embodiment, the cancer is brain cancer, leukemia, skin cancer,prostate cancer, thyroid cancer, colon cancer, lung cancer or sarcoma.In another embodiment the cancer is selected from the group consistingof glioma, glioblastoma multiforme, paraganglioma, suprantentorialprimordial neuroectodermal tumors, acute myeloid leukemia,myelodysplastic syndrome, chronic myelogenous leukemia, melanoma,breast, prostate, thyroid, colon, lung, central chondrosarcoma, centraland periosteal chondroma tumors, fibrosarcoma, and cholangiocarcinoma.

In one embodiment, disclosed herein is the use of a compound of generalformula (I), compound of general formula (I′), and/or compound ofgeneral formula (I″), or a pharmaceutically acceptable salt, hydrate,solvate or prodrug thereof, in a therapy. The compound may be useful ina method of inducing an immune response and/or inducing STING-dependenttype I interferon production in a subject, such as a mammal in need ofsuch inhibition, comprising administering an effective amount of thecompound to the subject.

In one embodiment, disclosed herein is a pharmaceutical compositioncomprising at least one compound of general formula (I), compound ofgeneral formula (I′), and/or compound of general formula (I″), or apharmaceutically acceptable salt, hydrate, solvate or prodrug thereof,for use in potential treatment to induce an immune response and/or toinduce STING-dependent type I interferon production.

In one embodiment, disclosed herein is the use of a compound of generalformula (I), compound of general formula (I′), and/or compound ofgeneral formula (I″), or a pharmaceutically acceptable salt, hydrate,solvate or prodrug thereof, in the manufacture of a medicament for thetreatment to induce an immune response and/or to induce STING-dependenttype I interferon production. In one embodiment, the disease or disorderto be treated is a cell proliferation disorder. In another embodiment,the cell proliferation disorder is cancer. In another embodiment, thecancer is brain cancer, leukemia, skin cancer, breast, prostate cancer,thyroid cancer, colon cancer, lung cancer, or sarcoma. In anotherembodiment, the cancer is glioma, glioblastoma multiforme,paraganglioma, suprantentorial primordial neuroectodermal tumors, acutemyeloid leukemia, myelodysplastic syndrome, chronic myelogenousleukemia, melanoma, breast, prostate, thyroid, colon, lung, centralchondrosarcoma, central and periosteal chondroma tumors, fibrosarcoma,and/or cholangiocarcinoma.

Compositions

The term “composition” as used herein is intended to encompass a dosageform comprising a specified compound in a specified amount, as well asany dosage form which results, directly or indirectly, from combinationof a specified compound in a specified amount.

Such term is intended to encompass a dosage form comprising a compoundof general formula (I), a compound of general formula (I′), and/or acompound of general formula (I″), or a pharmaceutically acceptable salt,hydrate, solvate, or prodrug thereof, and one or more pharmaceuticallyacceptable carriers or excipients. Accordingly, the compositions of thepresent disclosure encompass any composition made by admixing a compoundof the present disclosure and one or more pharmaceutically acceptablecarrier or excipients. By “pharmaceutically acceptable”, it is meant thecarriers or excipients are compatible with the compound disclosed hereinand with other ingredients of the composition.

For the purpose of inducing an immune response and/or inducing aSTING-dependent type I interferon production, the compounds, optionallyin the form of a salt, hydrate, solvate or prodrug, can be administeredby means that produces contact of the active agent with the agent's siteof action. They can be administered by conventional means available foruse in conjunction with pharmaceuticals, either as individualtherapeutic agents or in a combination of therapeutic agents. They canbe administered alone, but typically are administered with apharmaceutical carrier selected on the basis of the chosen route ofadministration and standard pharmaceutical practice.

In one embodiment, disclosed herein is a composition comprising acompound of general formula (I), a compound of general formula (I′),and/or a compound of general formula (I″), or a pharmaceuticallyacceptable salt thereof, and one or more pharmaceutically acceptablecarriers or excipients. The composition may be prepared and packaged inbulk form wherein an effective amount of a compound of the disclosurecan be extracted and then given to a subject, such as with powders orsyrups. Alternatively, the composition may be prepared and packaged inunit dosage form wherein each physically discrete unit contains aneffective amount of a compound of general formula (I), a compound ofgeneral formula (I′), and/or a compound of general formula (I″).

The compounds disclosed herein and a pharmaceutically acceptable carrieror excipient(s) will typically be formulated into a dosage form adaptedfor administration to a subject by a desired route of administration.For example, dosage forms include those adapted for (1) oraladministration, such as tablets, capsules, caplets, pills, troches,powders, syrups, elixirs, suspensions, solutions, emulsions, sachets,and cachets; and (2) parenteral administration, such as sterilesolutions, suspensions, and powders for reconstitution. Suitablepharmaceutically acceptable carriers or excipients will vary dependingupon the particular dosage form chosen. In addition, suitablepharmaceutically acceptable carriers or excipients may be chosen for aparticular function that they may serve in the composition. For example,certain pharmaceutically acceptable carriers or excipients may be chosenfor their ability to facilitate the production of uniform dosage forms.Certain pharmaceutically acceptable carriers or excipients may be chosenfor their ability to facilitate the production of stable dosage forms.Certain pharmaceutically acceptable carriers or excipients may be chosenfor their ability to facilitate the carrying or transporting of acompound disclosed herein, once administered to the subject, from oneorgan or portion of the body to another organ or another portion of thebody. Certain pharmaceutically acceptable carriers or excipients may bechosen for their ability to enhance patient compliance.

Suitable pharmaceutically acceptable excipients include the followingtypes of excipients: diluents, lubricants, binders, disintegrants,fillers, glidants, granulating agents, coating agents, wetting agents,solvents, co-solvents, suspending agents, emulsifiers, sweeteners,flavoring agents, flavor masking agents, coloring agents, anti-cakingagents, hemectants, chelating agents, plasticizers, viscosity increasingagents, antioxidants, preservatives, stabilizers, surfactants, andbuffering agents.

A skilled artisan possesses the knowledge and skill in the art to selectsuitable pharmaceutically acceptable carriers and excipients inappropriate amounts for the use in the disclosure. In addition, thereare a number of resources available to the skilled artisan, whichdescribe pharmaceutically acceptable carriers and excipients and may beuseful in selecting suitable pharmaceutically acceptable carriers andexcipients. Examples include Remington's Pharmaceutical Sciences (MackPublishing Company), The Handbook of Pharmaceutical Additives (GowerPublishing Limited), and The Handbook of Pharmaceutical Excipients (theAmerican Pharmaceutical Association and the Pharmaceutical Press).

The compositions of the disclosure are prepared using techniques andmethods known to those skilled in the art. Some methods commonly used inthe art are described in Remington's Pharmaceutical Sciences (MackPublishing Company).

In one embodiment, the disclosure is directed to a solid oral dosageform such as a tablet or capsule comprising an effective amount of acompound of the disclosure and a diluent or filler. Suitable diluentsand fillers include lactose, sucrose, dextrose, mannitol, sorbitol,starch (e.g., corn starch, potato starch, and pre-gelatinized starch),cellulose and its derivatives, (e.g., microcrystalline cellulose),calcium sulfate, and dibasic calcium phosphate. The oral solid dosageform may further comprise a binder. Suitable binders include starch(e.g., corn starch, potato starch, and pre-gelatinized starch) gelatin,acacia, sodium alginate, alginic acid, tragacanth, guar gum, povidone,and cellulose and its derivatives (e.g., microcrystalline cellulose).The oral solid dosage form may further comprise a disintegrant. Suitabledisintegrants include crospovidone, sodium starch glycolate,croscarmelose, alginic acid, and sodium carboxymethyl cellulose. Theoral solid dosage form may further comprise a lubricant. Suitablelubricants include stearic acid, magnesium stearate, calcium stearate,and talc.

Where appropriate, dosage unit formulations for oral administration canbe microencapsulated. The composition can also be prepared to prolong orsustain the release as, for example, by coating or embedding particulatematerial in polymers, wax, or the like.

The compounds disclosed herein may also be coupled with soluble polymersas targetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyrancopolymer,polyhydroxypropylmethacrylamidephenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds of thedisclosure may be coupled to a class of biodegradable polymers useful inachieving controlled release of a drug, for example polylactic acid,polepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters,polyacetals, polydihydropyrans, polycyanacrylates and cross-linked oramphipathic block copolymers of hydrogels.

In one embodiment, the disclosure is directed to a liquid oral dosageform. Oral liquids such as solution, syrups and elixirs can be preparedin dosage unit form so that a given quantity contains a predeterminedamount of a compound disclosed herein. Syrups can be prepared bydissolving the compound of the disclosure in a suitably flavored aqueoussolution; while elixirs are prepared through the use of a non-toxicalcoholic vehicle. Suspensions can be formulated by dispersing acompound disclosed herein in a non-toxic vehicle. Solubilizers andemulsifiers such as ethoxylated isostearyl alcohols and polyoxy ethylenesorbitol ethers, preservatives, flavor additives such as peppermint oilor other natural sweeteners or saccharin or other artificial sweetenersand the like can also be added.

In one embodiment, the disclosure is directed to compositions forparenteral administration. Compositions adapted for parenteraladministration include aqueous and non-aqueous sterile injectionsolutions which may contain anti-oxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient; and aqueous and non-aqueous sterile suspensions thatmay include suspending agents and thickening agents. The compositionsmay be presented in unit-dose or multi-dose containers, for examplesealed ampoules and vials, and may be stored in a freeze dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example water for injections, immediately prior touse. Extemporaneous injection solutions and suspensions may be preparedfrom sterile powders, granules and tablets.

Combinations

The compounds of general formula (I), compounds of general formula (I′),and/or compounds of general formula (I″), or pharmaceutically acceptablesalt, hydrate, solvate, or prodrug thereof, may be administered incombination with one or more additional therapeutic agents. Inembodiments, one or more a compound of general formula (I), compound ofgeneral formula (I′), or compound of general formula (I″), orpharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof,and the one or more additional therapeutic agents may be co-adminstered.The additional therapeutic agent(s) may be administered in a singledosage form with the compound of general formula (I), compound ofgeneral formula (I′), or compound of general formula (I″), orpharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof,or the additional therapeutic agent(s) may be administered in separatedosage form(s) from the dosage form containing the compound of generalformula (I), compound of general formula (I′), or compound of generalformula (I″), or pharmaceutically acceptable salt, hydrate, solvate, orprodrug thereof. The additional therapeutic agent(s) may be one or moreagents selected from the group consisting of STING agonist compounds,anti-viral compounds, antigens, adjuvants, anti-cancer agents, CTLA-4,LAG-3 and PD-1 pathway antagonists, lipids, peptides, cytotoxic agents,chemotherapeutic agents, immunomodulatory cell lines, checkpointinhibitors, vascular endothelial growth factor (VEGF) receptorinhibitors, topoisomerase II inhibitors, smoothen inhibitors, alkylatingagents, anti-tumor antibiotics, anti-metabolites, retinoids, andimmunomodulatory agents including but not limited to anti-cancervaccines. It will be understood the descriptions of the above additionaltherapeutic agents may be overlapping. It will also be understood thatthe treatment combinations are subject to optimization, and it isunderstood that the best combination to use of the compounds of generalformula (I), compounds of general formula (I′), or compounds of generalformula (I″) and one or more additional therapeutic agents will bedetermined based on the individual patient needs.

A compound disclosed herein may be used in combination with one or moreother active agents, including but not limited to, other anti-canceragents that are used in the prevention, treatment, control,amelioration, or reduction of risk of a particular disease or condition(e.g., cell proliferation disorders). In one embodiment, a compounddisclosed herein is combined with one or more other anti-cancer agentsfor use in the prevention, treatment, control amelioration, or reductionof risk of a particular disease or condition for which the compoundsdisclosed herein are useful. Such other active agents may beadministered, by a route and in an amount commonly used therefor,contemporaneously or sequentially with a compound of the presentdisclosure.

When a compound disclosed herein is used contemporaneously with one ormore other active agents, a composition containing such other activeagents in addition to the compound disclosed herein is contemplated.Accordingly, the compositions of the present disclosure include thosethat also contain one or more other active ingredients, in addition to acompound disclosed herein. A compound disclosed herein may beadministered either simultaneously with, or before or after, one or moreother therapeutic agent(s). A compound disclosed herein may beadministered separately, by the same or different route ofadministration, or together in the same pharmaceutical composition asthe other agent(s).

Products provided as combinations may include a composition comprising acompound of general formula (I), compound of general formula (I′), orcompound of general formula (I″), or pharmaceutically acceptable salt,hydrate, solvate, or prodrug thereof, and one or more other activeagent(s) together in the same pharmaceutical composition, or may includea composition comprising a compound of general formula (I), compound ofgeneral formula (I′), or compound of general formula (I″), orpharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof,and a composition comprising one or more other therapeutic agent(s) inseparate form, e.g. in the form of a kit or in any form designed toenable separate administration either concurrently or on separate dosingschedules.

The weight ratio of a compound disclosed herein to a second active agentmay be varied and will depend upon the effective dose of each agent.Generally, an effective dose of each will be used. Combinations of acompound disclosed herein and other active agents will generally also bewithin the aforementioned range, but in each case, an effective dose ofeach active agent should be used. In such combinations, the compounddisclosed herein and other active agents may be administered separatelyor in conjunction. In addition, the administration of one element may beprior to, concurrent to, or subsequent to the administration of otheragent(s).

In one embodiment, this disclosure provides a composition comprising acompound of general formula (I), compound of general formula (I′), orcompound of general formula (I″), or pharmaceutically acceptable salt,hydrate, solvate, or prodrug thereof, and at least one other therapeuticagent as a combined preparation for simultaneous, separate or sequentialuse in therapy. In one embodiment, the therapy is the treatment of acell proliferation disorder, such as cancer.

In one embodiment, the disclosure provides a kit comprising two or moreseparate pharmaceutical compositions, at least one of which contains acompound of general formula (I), compound of general formula (I′), orcompound of general formula (I″), or pharmaceutically acceptable salt,hydrate, solvate, or prodrug thereof. In one embodiment, the kitcomprises means for separately retaining said compositions, such as acontainer, divided bottle, or divided foil packet. An example of such akit is a blister pack, as typically used for the packaging of tablets,capsules, and the like.

A kit of this disclosure may be used for administration of differentdosage forms, for example, oral and parenteral, for administration ofthe separate compositions at different dosage intervals, or fortitration of the separate compositions against one another. To assistwith compliance, a kit of the disclosure typically comprises directionsfor administration.

Disclosed herein is a use of a compound of general formula (I), compoundof general formula (I′), or compound of general formula (I″), orpharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof,for treating a cell proliferation disorder, wherein the medicament isprepared for administration with another active agent. The disclosurealso provides the use of another active agent for treating a cellproliferation disorder, wherein the medicament is administered with acompound of general formula (I).

The disclosure also provides the use of a compound of general formula(I), compound of general formula (I′), or compound of general formula(I″), or pharmaceutically acceptable salt, hydrate, solvate, or prodrugthereof, for treating a cell proliferation disorder, wherein the patienthas previously (e.g., within 24 hours) been treated with another activeagent. The disclosure also provides the use of another therapeutic agentfor treating a cell proliferation disorder, wherein the patient haspreviously (e.g., within 24 hours) been treated with a compound ofgeneral formula (I), compound of general formula (I′), or compound ofgeneral formula (I″), or pharmaceutically acceptable salt, hydrate,solvate, or prodrug thereof. The second agent may be applied a week,several weeks, a month, or several months after the administration of acompound disclosed herein.

STING agonist compounds that may be used in combination with thecompounds of general formula (I), compounds of general formula (I′), andcompounds of general formula (I″) disclosed herein include but are notlimited to cyclic di-nucleotide compounds.

Anti-viral compounds that may be used in combination with the compoundsof general formula (I), compounds of general formula (I′), and compoundsof general formula (I″) disclosed herein include hepatitis B virus (HBV)inhibitors, hepatitis C virus (HCV) protease inhibitors, HCV polymeraseinhibitors, HCV NS4 Å inhibitors, HCV NS5 Å inhibitors, HCV NS5binhibitors, and human immunodeficiency virus (HIV) inhibitors.

Antigens and adjuvants that may be used in combination with thecompounds of general formula (I), compounds of general formula (I′), andcompounds of general formula (I″) disclosed herein include B7costimulatory molecule, interleukin-2, interferon-y, GM-CSF, CTLA-4antagonists, OX-40/OX-40 ligand, CD40/CD40 ligand, sargramostim,levamisol, vaccinia virus, Bacille Calmette-Guerin (BCG), liposomes,alum, Freund's complete or incomplete adjuvant, detoxified endotoxins,mineral oils, surface active substances such as lipolecithin, pluronicpolyols, polyanions, peptides, and oil or hydrocarbon emulsions.Adjuvants, such as aluminum hydroxide or aluminum phosphate, can beadded to increase the ability of the vaccine to trigger, enhance, orprolong an immune response. Additional materials, such as cytokines,chemokines, and bacterial nucleic acid sequences, like CpG, a toll-likereceptor (TLR) 9 agonist as well as additional agonists for TLR 2, TLR4, TLR 5, TLR 7, TLR 8, TLR9, including lipoprotein, LPS,monophosphoryllipid A, lipoteichoic acid, imiquimod, resiquimod, and inaddition retinoic acid-inducible gene I (RIG-I) agonists such as polyI:C, used separately or in combination with the described compositionsare also potential adjuvants.

CLTA-4 and PD-1 pathways are important negative regulators of immuneresponse. Activated T-cells upregulate CTLA-4, which binds onantigen-presenting cells and inhibits T-cell stimulation, IL-2 geneexpression, and T-cell proliferation; these anti-tumor effects have beenobserved in mouse models of colon carcinoma, metastatic prostate cancer,and metastatic melanoma. PD-1 binds to active T-cells and suppressesT-cell activation; PD-1 antagonists have demonstrated anti-tumor effectsas well. CTLA-4 and PD-1 pathway antagonists that may be used incombination with the compounds of general formula (I) disclosed hereininclude ipilimumab, tremelimumab, nivolumab, pembrolizumab, CT-011,AMP-224, and MDX-1106.

“PD-1 antagonist” or “PD-1 pathway antagonist” means any chemicalcompound or biological molecule that blocks binding of PD-L1 expressedon a cancer cell to PD-1 expressed on an immune cell (T-cell, B-cell, orNKT-cell) and preferably also blocks binding of PD-L2 expressed on acancer cell to the immune-cell expressed PD-1. Alternative names orsynonyms for PD-1 and its ligands include: PDCD1, PD1, CD279, and SLEB2for PD-1; PDCD1 L1, PDL1, B7H1, B7-4, CD274, and B7-H for PD-L1; andPDCD1 L2, PDL2, B7-DC, Btdc, and CD273 for PD-L2. In any of thetreatment method, medicaments and uses of the present disclosure inwhich a human individual is being treated, the PD-1 antagonist blocksbinding of human PD-L1 to human PD-1, and preferably blocks binding ofboth human PD-L1 and PD-L2 to human PD-1. Human PD-1 amino acidsequences can be found in NCBI Locus No.: NP_005009. Human PD-L1 andPD-L2 amino acid sequences can be found in NCBI Locus No.: NP_054862 andNP_079515, respectively.

PD-1 antagonists useful in any of the treatment method, medicaments anduses of the present disclosure include a monoclonal antibody (mAb), orantigen binding fragment thereof, which specifically binds to PD-1 orPD-L1, and preferably specifically binds to human PD-1 or human PD-L1.The mAb may be a human antibody, a humanized antibody, or a chimericantibody and may include a human constant region. In some embodiments,the human constant region is selected from the group consisting of IgG1,IgG2, IgG3, and IgG4 constant regions, and in preferred embodiments, thehuman constant region is an IgG1 or IgG4 constant region. In someembodiments, the antigen binding fragment is selected from the groupconsisting of Fab, Fab′-SH, F(ab′)₂, scFv, and Fv fragments.

Examples of mAbs that bind to human PD-1, and useful in the treatmentmethod, medicaments and uses of the present disclosure, are described inU.S. Pat. Nos. 7,488,802, 7,521,051, 8,008,449, 8,354,509, and8,168,757, PCT International Patent Application Publication Nos.WO2004/004771, WO2004/072286, and WO2004/056875, and U.S. PatentApplication Publication No. US2011/0271358.

Examples of mAbs that bind to human PD-L1, and useful in the treatmentmethod, medicaments and uses of the present disclosure, are described inPCT International Patent Application Nos. WO2013/019906 andWO2010/077634 A1 and in U.S. Pat. No. 8,383,796. Specific anti-humanPD-L1 mAbs useful as the PD-1 antagonist in the treatment method,medicaments and uses of the present disclosure include MPDL3280 Å,BMS-936559, MEDI4736, MSB0010718C, and an antibody that comprises theheavy chain and light chain variable regions of SEQ ID NO:24 and SEQ IDNO:21, respectively, of WO2013/019906.

Other PD-1 antagonists useful in any of the treatment method,medicaments, and uses of the present disclosure include animmune-adhesion that specifically binds to PD-1 or PD-L1, and preferablyspecifically binds to human PD-1 or human PD-L1, e.g., a fusion proteincontaining the extracellular or PD-1 binding portion of PD-L1 or PD-L2fused to a constant region such as an Fc region of an immunoglobulinmolecule. Examples of immune-adhesion molecules that specifically bindto PD-1 are described in PCT International Patent ApplicationPublication Nos. WO2010/027827 and WO2011/066342. Specific fusionproteins useful as the PD-1 antagonist in the treatment method,medicaments, and uses of the present disclosure include AMP-224 (alsoknown as B7-DCIg), which is a PD-L2-FC fusion protein and binds to humanPD-1.

Examples of cytotoxic agents that may be used in combination with thecompounds of general formula (I), compounds of general formula (I′), andcompounds of general formula (I″), or pharmaceutically acceptable salts,hydrates, solvates, or prodrugs thereof, disclosed herein include, butare not limited to, arsenic trioxide (sold under the tradenameTRISENOX®), asparaginase (also known as L-asparaginase, and ErwiniaL-asparaginase, sold under the tradenames ELSPAR® and KIDROLASE®).

Chemotherapeutic agents that may be used in combination with thecompounds of general formula (I), compounds of general formula (I′), andcompounds of general formula (I″), or pharmaceutically acceptable salts,hydrates, solvates, or prodrugs thereof, disclosed herein includeabiraterone acetate, altretamine, anhydrovinblastine, auristatin,bexarotene, bicalutamide, BMS 184476,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide,bleomycin,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-proly-1-Lproline-tbutylamide,cachectin, cemadotin, chlorambucil, cyclophosphamide,3′,4′-didehydro-4′deoxy-8′-norvin-caleukoblastine, docetaxol, doxetaxel,cyclophosphamide, carboplatin, carmustine, cisplatin, cryptophycin,cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin,daunorubicin, decitabine dolastatin, doxorubicin (adriamycin),etoposide, 5-fluorouracil, finasteride, flutamide, hydroxyurea andhydroxyureataxanes, ifosfamide, liarozole, lonidamine, lomustine (CCNU),MDV3100, mechlorethamine (nitrogen mustard), melphalan, mivobulinisethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate,taxanes, nilutamide, nivolumab, onapristone, paclitaxel, pembrolizumab,prednimustine, procarbazine, RPR109881, stramustine phosphate,tamoxifen, tasonermin, taxol, tretinoin, vinblastine, vincristine,vindesine sulfate, and vinflunine.

Examples of vascular endothelial growth factor (VEGF) receptorinhibitors include, but are not limited to, bevacizumab (sold under thetrademark AVASTIN by Genentech/Roche), axitinib (described in PCTInternational Patent Publication No. WO1/002369), Brivanib Alaninate((S)—((R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate,also known as BMS-582664), motesanib(N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridinylmethyl)amino]-3-pyridinecarboxamide.and described in PCT International Patent Application Publication No.WO02/068470), pasireotide (also known as SO 230, and described in PCTInternational Patent Publication No. WO02/010192), and sorafenib (soldunder the tradename NEXAVAR).

Examples of topoisomerase II inhibitors, include but are not limited to,etoposide (also known as VP-16 and Etoposide phosphate, sold under thetradenames TOPOSAR, VEPESID, and ETOPOPHOS), and teniposide (also knownas VM-26, sold under the tradename VUMON).

Examples of alkylating agents, include but are not limited to,5-azacytidine (sold under the trade name VIDAZA), decitabine (sold underthe trade name of DECOGEN), temozolomide (sold under the trade namesTEMODAR and TEMODAL by Schering-Plough/Merck), dactinomycin (also knownas actinomycin-D and sold under the tradename COSMEGEN), melphalan (alsoknown as L-PAM, L-sarcolysin, and phenylalanine mustard, sold under thetradename ALKERAN), altretamine (also known as hexamethylmelamine (HMM),sold under the tradename HEXALEN), carmustine (sold under the tradenameBCNU), bendamustine (sold under the tradename TREANDA), busulfan (soldunder the tradenames BUSULFEX® and MYLERAN®), carboplatin (sold underthe tradename PARAPLATIN®), lomustine (also known as CCNU, sold underthe tradename CEENU®), cisplatin (also known as CDDP, sold under thetradenames PLATINOL® and PLATINOL®-AQ), chlorambucil (sold under thetradename LEUKERAN®), cyclophosphamide (sold under the tradenamesCYTOXAN® and NEOSAR®), dacarbazine (also known as DTIC, DIC andimidazole carboxamide, sold under the tradename DTIC-DOME®), altretamine(also known as hexamethylmelamine (HMM) sold under the tradenameHEXALEN®), ifosfamide (sold under the tradename IFEX®), procarbazine(sold under the tradename MATULANE®), mechlorethamine (also known asnitrogen mustard, mustine and mechloroethamine hydrochloride, sold underthe tradename MUSTARGEN®), streptozocin (sold under the tradenameZANOSAR®), thiotepa (also known as thiophosphoamide, TESPA and TSPA, andsold under the tradename THIOPLEX®.

Examples of anti-tumor antibiotics include, but are not limited to,doxorubicin (sold under the tradenames ADRIAMYCIN® and RUBEX®),bleomycin (sold under the tradename LENOXANE®), daunorubicin (also knownas dauorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride,sold under the tradename CERUBIDINE®), daunorubicin liposomal(daunorubicin citrate liposome, sold under the tradename DAUNOXOME®),mitoxantrone (also known as DHAD, sold under the tradename NOVANTRONE®),epirubicin (sold under the tradename ELLENCE™), idarubicin (sold underthe tradenames IDAMYCIN®, IDAMYCIN PFS®), and mitomycin C (sold underthe tradename MUTAMYCIN®).

Examples of anti-metabolites include, but are not limited to, claribine(2-chlorodeoxyadenosine, sold under the tradename LEUSTATIN®),5-fluorouracil (sold under the tradename ADRUCIL®), 6-thioguanine (soldunder the tradename PURINETHOL®), pemetrexed (sold under the tradenameALIMTA®), cytarabine (also known as arabinosylcytosine (Ara-C), soldunder the tradename CYTOSAR-U®), cytarabine liposomal (also known asLiposomal Ara-C, sold under the tradename DEPOCYT™), decitabine (soldunder the tradename DACOGEN®), hydroxyurea (sold under the tradenamesHYDREA®, DROXIA™ and MYLOCEL™), fludarabine (sold under the tradenameFLUDARA®), floxuridine (sold under the tradename FUDR®), cladribine(also known as 2-chlorodeoxyadenosine (2-CdA) sold under the tradenameLEUSTATIN™), methotrexate (also known as amethopterin, methotrexatesodium (MTX), sold under the tradenames RHEUMATREX® and TREXALL™), andpentostatin (sold under the tradename NIPENT®).

Examples of retinoids include, but are not limited to, alitretinoin(sold under the tradename PANRETIN®), tretinoin (all-trans retinoicacid, also known as ATRA, sold under the tradename VESANOID®),Isotretinoin (13-c/s-retinoic acid, sold under the tradenames ACCUTANE®,AMNESTEEM®, CLARAVIS®, CLARUS®, DECUTAN®, ISOTANE®, IZOTECH®, ORATANE®,ISOTRET®, and SOTRET®), and bexarotene (sold under the tradenameTARGRETIN®).

Activity: STING Biochemical [3H]cGAMP Competition Assay

The individual compounds described in the Examples herein are defined asSTING agonists by demonstrating binding to the STING protein with anEC₅₀ of 20 uM or less in the STING Biochemical [3H]cGAMP CompetitionAssay and demonstrating interferon production with a 20% or greaterluminescence induction at 30 uM in the IFN-β secretion in the THP1 cellassay.

The ability of compounds to bind STING is quantified by the ability tocompete with tritiated cGAMP ligand for human STING receptor membraneusing a radioactive filter-binding assay. The binding assay employsSTING receptor obtained from Hi-Five cell membranes overexpressingfull-length HAQ STING prepared in-house and tritiated cGAMP ligand alsopurified in-house.

Abbreviations

-   ¹H-NMR Proton nuclear magnetic resonance spectroscopy-   ¹⁹F-NMR ¹⁹F nuclear magnetic resonance spectroscopy-   ³¹P-NMR ³¹P nuclear magnetic resonance spectroscopy-   Å Angstrom-   A^(Bz) 6-N-benzoyladenine-   aq Aqueous-   Ar Argon-   ATP Adenosine 5′-triphosphate-   Bz Benzoyl-   CD₃OD Deuterium-enriched methyl alcohol, deuterium-enriched methanol-   CHCl₃ Trichloromethane-   Ci Curie, a non-standard unit of radioactivity; 1Ci=3.7×10¹⁰Bq,    where Bq is Becquerel, the SI unit of radioactivity, equivalent to 1    disintegration per second (dps)-   CO₂ Carbon dioxide-   d Doublet-   d Day(s)-   D₂O Deuterium-enriched water-   DCA Dichloroacetic acid-   DCM, CH₂Cl₂ Dichloromethane-   ddd Doublet of doublet of doublet-   ddt Doublet of doublet of triplet-   DDTT    (E)-N,N-dimethyl-N′-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide-   DMF N,N-dimethylformamide-   DMOCP 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphineane 2-oxide-   DMSO Dimethyl sulfoxide-   DMTr 4,4′-dimethoxytrityl-   DMTrCl 4,4′-dimethoxytrityl chloride-   dq Doublet of quartet-   EC₅₀ half maximal effective concentration, concentration of a drug,    antibody or toxicant that induces a response halfway between the    baseline and maximum after a specified exposure time-   eq Equivalents-   ES Electron spray-   Et Ethyl-   Et₂O Diethyl ether-   Et₃ SiH Triethylsilane-   EtOAc Ethyl acetate-   EtOH Ethyl alcohol, ethanol-   g Gram-   GTP Guanosine 5′-triphosphate-   h Hour-   H₂O Water-   HEPES 2-[4-(2-hydroxyethyl)piperazin 1-yl]ethanesulfonic acid, a    zwitterionic organic chemical buffering agent-   hept Heptet-   Hex Hexanes-   HF-Pyr Hydrogen fluoride-pyridine complex-   HPLC High performance liquid chromatography-   Hz Hertz-   ITP Inosine 5′-triphosphate-   J NMR Coupling constant-   LCMS Liquid chromatography-mass spectroscopy-   m Multiplet-   M Molar, moles per liter-   mCi Millicurie-   Me Methyl-   MeCN Acetonitrile-   MeNH₂ Methylamine-   mg Milligram-   MgCl₂ Magnesium chloride-   MHz Megahertz-   min Minute(s)-   mL, ml Milliliter-   mM Millimole per liter-   mmol Millimole-   MOI Multiplicity of infection-   MPLC Medium pressure liquid chromatography-   MTBE Methyl t-butyl ether, methyl tertiary butyl ether-   Na₂SO₄ Sodium sulfate-   NaCl Sodium chloride-   NaHCO₃ Sodium bicarbonate-   NaHSO₃ Sodium bisulfite-   NaOH Sodium hydroxide-   ng Nanogram(s)-   NH₄HCO₃ Ammonium bicarbonate-   NH₄OH Ammonium hydroxide-   nL Nanoliter-   nm Nanometer-   nM Nanomolar-   P₂O₅ Phosphorus pentoxide-   Py Pyridine-   q Quartet-   RPM, rpm Revolutions per minute-   RT, rt Room temperature, approximately 25° C.-   s Singlet-   sat Saturated-   t Triplet-   TB S t-Butyldimethylsilyl-   TMA Trimethylamine-   TEA, Et₃N Triethylamine-   TFA Trifluoroacetic acid-   TLC Thin layer chromatography-   TMSCl Trimethylsilyl chloride-   T_(R) Retention time-   TrisCl Tris(hydroxymethyl)aminomethane hydrochloride-   v/v Volume/volume-   λ_(em) Emission wavelength-   λ_(ex) Excitation wavelength-   μg Microgram-   μL, uL Microliter-   μM, uM Micromolar

Preparation 1:N-(3-((2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)-methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)benzamide

Step 1:(2R,3R,4S,5R)-5-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((benzoyloxy)methyl)-4-fluorotetrahydrofuran-3-ylbenzoate

To a mixture of 3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-amine (2.36 g, 17.4mmol) in NMP (50 ml) was added NaH (60%, 0.744 g, 18.6 mmol). Themixture was vigorously stirred and after 1 h, generation of bubbles hadcompletely ceased. The mixture was added to((2R,3R,4S,5R)-3-(benzoyloxy)-5-bromo-4-fluorotetrahydrofuran-2-yl)methylbenzoate (neat, 5.25 g, 12.4 mmol) in one portion. The reaction wasstirred for 18 h. LCMS showed several peaks with the desired mass(m/e=479). EtOAc (70 mL) and water (70 mL) were added to the reaction.Layers were separated, and the organic layer was washed with halfsaturated brine (3×10 mL) and brine (1×10 mL), dried (MgSO₄), andconcentrated. The crude was purified via silica column eluting with 0 to50% EtOAc in Hex to give the product. LCMS (ES, m/z): 479.3 [M+H]⁺.¹H-NMR (500 MHz, DMSO-d₆): δ 8.62 (s, 1H), 8.34 (s, 1H), 8.28 (s, 1H),8.10-8.04 (m, 2H), 7.97-7.90 (m, 2H), 7.77-7.69 (m, 1H), 7.67-7.55 (m,3H), 7.49-7.42 (m, 2H), 6.97 (dd, J=6.5, 3.1 Hz, 1H), 6.49 (dt, J=17.6,6.9 Hz, 1H), 6.16 (dt, J=56, 6.6 Hz, 1H), 4.76-4.62 (m, 3H).

Step 2.(2R,3R,4S,5R)-5-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol

To a solution of(2R,3R,4S,5R)-5-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((benzoyloxy)methyl)-4-fluorotetrahydrofuran-3-ylbenzoate (2.00 g, 4.18 mmol) in pyridine (10 mL) at was added NH₃ inMeOH (7N, 20 mL, 140 mmol). It was stirred for 48 h. LCMS showedcompletion of the reaction (m/e=271). It was concentrated and purifiedby silica column chromatography eluting with 10% MeOH in CH₂Cl₂ to givethe desired product. LCMS (ES, m/z): 271.1 [M+H]⁺. ¹H-NMR (500 MHz,DMSO-d6): δ 8.54 (s, 1H), 8.33 (s, 1H), 8.22 (s, 1H), 6.73 (dd, J=6.5,2.6 Hz, 1H), 6.00 (d, J=5.4 Hz, 1H), 5.51 (ddd, J=53, 7.2, 6.5 Hz, 1H),4.93 (t, J=5.8 Hz, 1H), 4.86-4.74 (m, 1H), 3.91-3.83 (m, 1H), 3.77-3.61(m, 2H).

Step 3:N-(3-((2R,3S,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)benzamide

To a solution of(2R,3R,4S,5R)-5-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol(1.34 g, 4.96 mmol) in pyridine (30 mL) at 0° C. was added TMSCl (1.46mL, 11.4 mmol). It was warmed to rt and stirred for 1 h. Then, it wasrecooled to 0° C. and BzCl (0.921 mL, 7.93 mmol) was added dropwise. Thereaction was slowly warmed to rt over 2 h. LCMS showed completion ofreaction (m/e=375, 479). Water (3 mL) was added. It was cooled to 0° C.and NH₃ in MeOH (7N, 2.8 mL, 20 mmol) was added. After 1 h, the reactionmixture was concentrated. It was purified by silica columnchromatography eluting with 0 to 10% MeOH in CH₂Cl₂ to give the product.LCMS (ES, m/z): 375.2 [M+H]⁺. ¹H-NMR (500 MHz, DMSO-d₆): δ 11.95 (s,1H), 8.98 (s, 1H), 8.10 (d, J=7.6 Hz, 2H), 7.73-7.66 (m, 1H), 7.59 (t,J=7.7 Hz, 2H), 6.91 (d, J=6.2 Hz, 1H), 6.06 (d, J=5.6 Hz, 1H), 5.59 (t,J=53, 6.8 Hz, 1H), 4.90 (t, J=5.8 Hz, 1H), 4.82 (dq, J=19.8, 7.0 Hz,1H), 3.92 (td, J=7.6, 2.9 Hz, 1H), 3.75 (ddd, J=12.1, 5.6, 3.0 Hz, 1H),3.66 (dt, J=12.0, 6.6 Hz, 1H).

Step 4:N-(3-((2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)benzamide

To a solution ofN-(3-((2R,3S,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)benzamide(1.25 g, 3.34 mmol) in pyridine (15 mL) at 0° C. was added DMTrCl (1.58g, 4.68 mmol). It was stirred at rt for 1 h. LCMS showed a peak with thedesired mass (m/e=677). It was partly concentrated (to 5 mL), and EtOAc(20 mL) and water (10 mL) were added. Layers were separated, and the aqlayer was extracted with EtOAc (2×10 mL). The combined organics werewashed with brine (5 mL), dried (MgSO₄), concentrated and purified bysilica column chromatography eluting with 0 to 60% EtOAc in Hex to givethe product. LCMS (ES, m/z): 675.5 [M−H]⁻. ¹H-NMR (500 MHz, DMSO-d₆): δ8.13-8.07 (m, 2H), 7.69 (t, J=7.4 Hz, 1H), 7.59 (t, J=7.6 Hz, 2H),7.35-7.29 (m, 2H), 7.23-7.10 (m, 6H), 6.97 (d, J=6.5 Hz, 1H), 6.81-6.74(m, 2H), 6.74-6.67 (m, 2H), 6.07 (d, J=5.7 Hz, 1H), 5.62 (dt, J=53, 7.0Hz, 1H), 4.91-4.79 (m, 1H), 4.15-4.07 (m, 1H), 3.69 (s, 3H), 3.67 (s,3H), 3.44 (dd, J=10.4, 8.0 Hz, 1H), 3.21 (dd, J=10.3, 2.4 Hz, 1H).

Preparation 2:(2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite

Step 1:(2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite

To a solution of 3-((bis(diisopropylamino)phosphino)oxy)propanenitrile(8.02 g, 26.6 mmol) in ACN (90 mL) at rt was added pyridin-1-ium2,2,2-trifluoroacetate (3.85 g, 19.95 mmol) and a solution ofN-(3-((2R,3 S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)benzamide(9 g, 13.30 mmol) in ACN (90 mL). The resulting mixture was stirred for1 h. Then, it was concentrated and the residue was dissolved in CH₂Cl₂(1000 mL). It was washed with aq NaHCO₃ (1%, 2×300 mL), water (300 mL)and brine (300 mL), dried (Na₂SO₄), concentrated, and purified byreverse phase (C18) chromatography eluting with 0 to 95% ACN in water togive the product. LCMS (ES, m/z): 877.5 [M+H]⁺. ¹H-NMR: (400 MHz,DMSO-d₆): δ 12.01 (s, 1H), 8.92 (s, 1H), 8.11 (d, J=7.6 Hz, 2H), 7.66(dt, J=42.3, 7.5 Hz, 3H), 7.32 (td J=7.2, 6.6, 2.9 Hz, 2H), 7.22-7.00(m, 9H), 6.83-6.63 (m, 4H), 5.86 (ddt, J=52.8, 17.6, 6.9 Hz, 1H), 5.16(td, J=17.7, 17.2, 8.8 Hz, 1H), 3.78-3.63 (m, 7H), 3.59-3.35 (m, 5H),2.74 (t, J=5.9 Hz, 1H), 2.63 (t, J=5.9 Hz, 1H), 1.23-0.99 (m, 10H), 0.91(d, J=6.7 Hz, 2H). ³¹P-NMR: (162 MHz, DMSO-d₆): δ 150.26, 149.60 (2 s,1P).

Preparation 3.N-(9-((2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

Step 1:N-(9-((2R,3S,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

To a suspension of 2-amino-9-((2R,3S,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one(Carbosynth catalog # ND10826, 1.50 g, 5.26 mmol) in pyridine (30 mL) at0-5° C. was added TMSCl (2.86 g, 26.3 mmol), and the mixture was stirredat rt for 30 min. Then, isobutyric anhydride (2.50 g, 15.8 mmol) wasadded dropwise, and it was stirred for an additional for 2 h. Then, MeOH(5.3 mL) was added. After 5 min, NH₄OH (10.5 mL) was added dropwise andstirring was continued for 30 min. The reaction mixture was concentratedunder reduced pressure, and MeOH (2 mL) in CH₂Cl₂ (18 mL) was added tothe residue. Insolubles were filtered off, and the filtrate wasconcentrated and purified by flash column chromatography with 2-10% MeOHin CH₂Cl₂ to give the product. LCMS (ES, m/z): 356.1 [M+H]⁺. ¹H-NMR:(400 MHz, DMSO-d₆): δ 12.11 (s, 1H), 11.68 (s, 1H), 8.28 (s, 1H), 5.98(d, J=6.1 Hz, 1H), 5.85 (d, J=8.0 Hz, 1H), 5.24 (t, J=5.4 Hz, 1H), 5.14(d, J=4.1 Hz, 0.5H), 5.01 (d, J=4.2 Hz, 0.5H), 4.87-4.69 (m, 1H), 4.26(t, J=4.4 Hz, 0.5H), 4.19 (t, J=4.4 Hz, 0.5H), 3.61 (t, J=4.9 Hz, 2H),2.77 (hept, J=6.8 Hz, 1H), 1.13 (d, J=6.7 Hz, 6H). ¹⁹F-NMR: (376 MHz,DMSO-d₆): δ −197.5 (s).

Step 2:N-(9-((2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

N-(9-((2R,3S,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide(1.30 g, 3.66 mmol) was co-evaporated with pyridine (3×10 mL) andre-dissolved in pyridine (26 mL). To the solution at 0-5° C. was addedDMTrCl (1.36 g, 4.02 mmol). It was stirred at rt for 3 h and then,concentrated. CH₂Cl₂ (40 mL, with 1% Et₃N) was added, and it was washedwith sat aq NaHCO₃ (15 mL), water (10 mL) and brine (10 mL). The organicsolution was dried (Na₂SO₄), concentrated and purified by silica gelcolumn chromatography using 0-10% MeOH in CH₂Cl₂ (1% Et₃N) to give theproduct. LCMS (ES, m/z): 656.2 [M−H]⁻. ¹H-NMR: (400 MHz, DMSO-d₆): δ12.10 (s, 1H), 11.61 (s, 1H), 8.14 (s, 1H), 7.40-7.31 (m, 2H), 7.31-7.19(m, 7H), 6.89-6.78 (m, 4H), 6.08 (d, J=6.1 Hz, 1H), 5.87 (d, J=7.3 Hz,1H), 5.23 (dd, J=4.1, 1.8 Hz, 0H), 5.10 (d, J=4.4 Hz, 0H), 4.96 (dq,J=22.4, 5.9 Hz, 1H), 4.30 (dt, J=26.1, 4.6 Hz, 1H), 3.74 (d, J=1.1 Hz,6H), 3.39 (dd, J=10.6, 5.7 Hz, 1H), 3.22 (dd, J=10.6, 3.8 Hz, 1H), 2.76(p, J=6.8 Hz, 1H), 1.13 (d, J=6.8 Hz, 6H). ¹⁹F-NMR: (376 MHz, DMSO-d₆):δ −198.1 (s, 1F).

The product of Preparation 3 may optionally be treated according to theprocedures of Preparation 22, Steps 4 and 5 (below), to afford(2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylhydrogen phosphonate. LCMS (ES, m/z): 720 [M−H]⁻.

Preparation 4:N-(3-((2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

Step 1:((2R,3R,4S,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-4-(benzoyloxy)-3-fluorotetrahydrofuran-2-yl)methylbenzoate

To a suspension of 8-azaguanine (5.14 g, 33.8 mmol) in anhydrous CH₃CN(100 mL) at rt was added dropwise (E)-trimethylsilylN-(trimethylsilyl)acetimidate (16.53 mL, 67.6 mmol), then the mixturewas stirred at 70° C. for 2 h. The reaction was cooled to rt, and asolution of((2R,3R,4S)-5-acetoxy-4-(benzoyloxy)-3-fluorotetrahydrofuran-2-yl)methylbenzoate (6.8 g, 16.90 mmol) in anhydrous CH₃CN (20 mL) was addedfollowed by dropwise addition of tin(IV) chloride (67.6 mL, 67.6 mmol).The homogeneous solution was stirred at 70° C. for 2 h. The reaction wascooled to rt and concentrated. The residue was dissolved in EtOAc (1000mL) and neutralized by pouring into sat aq NaHCO₃ (500 mL). The organiclayer was separated, and the aq layer was extracted with EtOAc (4×500mL). The organic layers were combined and washed with water (3×700 mL),brine, dried over anhydrous sodium sulfate, filtered, and concentratedto afford title compound without further purification. LCMS (ES, m/z):495.3 [M+H]⁺.

Step 2:((2R,3R,4S,5R)-4-(benzoyloxy)-3-fluoro-5-(5-isobutyramido-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl)methylbenzoate

To a solution of((2R,3R,4S,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-4-(benzoyloxy)-3-fluorotetrahydrofuran-2-yl)methylbenzoate (8 g, 16.18 mmol) from Step 1 in anhydrous DMA (40 mL) at rtwas added dropwise isobutyric anhydride (4.02 mL, 24.27 mmol). Themixture was stirred at 140° C. for 4 h. The reaction was cooled anddiluted with EtOAc (600 mL), washed with sat aq NH₄Cl (4×500 mL), brine,dried over anhydrous sodium sulfate, filtered and concentrated. Thecrude product was purified by MPLC (220 g silica gel, eluting with agradient of 100% hexanes to 100% ethyl acetate) to afford((2R,3R,4S,5R)-4-(benzoyloxy)-3-fluoro-5-(5-isobutyramido-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl)methylbenzoate. LCMS (ES, m/z): 565.3 [M+H]⁺.

Step 3:N-(3-((2R,3S,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

To a solution of((2R,3R,4S,5R)-4-(benzoyloxy)-3-fluoro-5-(5-isobutyramido-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl)methylbenzoate (6 g, 10.63 mmol) in THF (20 mL), CH₃OH (16 mL), and water (4mL) at 0° C. was added 5N aqueous NaOH (4.89 mL, 24.45 mmol) and stirredfor 1 h. The reaction was neutralized with formic acid (1.223 mL, 31.9mmol). The solvent was removed, and the residue was purified by MPLC(120 g, silica gel, eluting with a gradient of 100% CH₂Cl₂ to 20%CH₃OH/CH₂Cl₂) to afford N-(3-((2R,3S,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide.LCMS (ES, m/z): 357.2 [M+H]⁺.

Step 4:N-(3-((2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

To a solution ofN-(3-((2R,3S,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide(3 g, 8.42 mmol) in anhydrous pyridine (40 mL) at 0° C. was added4,4′-dimethoxytrityl chloride (3.42 g, 10.10 mmol). The ice bath wasremoved, and the reaction mixture was allowed to reach RT and wasstirred for 2 h. The mixture was diluted with EtOAc (400 mL), washedwith sat aq NaHCO₃ (100 mL), water (3×100 mL), brine, and dried overanhydrous sodium sulfate, filtered and concentrated. The crude productwas purified by MPLC (120 g silica gel, eluting with a gradient of 100%CH₂Cl₂ to 15% CH₃OH/CH₂CH₂ to affordN-(3-((2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide.LCMS (ES, m/z): 659.3 [M+H]⁺.

Preparation 5:N-(3-((2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

N-(3-((2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramidewas prepared according to procedures analogous to those described forPreparation 4 using the appropriately substituted ribose in Step 1. LCMS(ES, m/z): 659.4 [M+H]⁺.

Preparation 6:N-(3-((2R,3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-hydroxytetrahydrofuran-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

N-(3-((2R,3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-hydroxytetrahydrofuran-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramidewas prepared according to procedures analogous to those described forPreparation 4 using the appropriately substituted ribose in Step 1. LCMS(ES, m/z): 641.2 [M+H]⁺.

Preparation 7:3-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-2-fluoro-β-D-arabinofuranosyl}-N-(phenylcarbonyl)-3H-imidazo[4,5-b]pyridin-7-amine

Step 1:3-[2-deoxy-2-fluoro-3,5-bis-O-(phenylcarbonyl)-β-D-arabinofuranosyl]-7-nitro-3H-imidazo[4,5-b]pyridine

To a stirred mixture of freshly ground KOH (308 mg, 5.48 mmol) inacetonitrile (50 mL) was added tris(2-(2-methoxyethoxy)ethyl)amine(0.070 mL, 0.219 mmol). The reaction mixture was aged for 15 min atambient temperature followed by addition of7-nitro-3H-imidazo[4,5-b]pyridine (600 mg, 3.66 mmol) in a singleportion. The resulting mixture was stirred at ambient temperature for 15min. A solution of2-deoxy-2-fluoro-3,5-bis-O-(phenylcarbonyl)-α-D-arabinofuranosyl bromide(1700 mg, 4.02 mmol) in acetonitrile (10 mL) was added dropwise and theresulting mixture was vigorously stirred at RT for 17 h. The reactionmixture was diluted with sat aq ammonium chloride (80 mL) and extractedwith DCM (3×150 mL). The organic extracts were combined, dried oversodium sulphate and concentrated under reduced pressure. The resultingresidue was purified by column chromatography on silica gel; 120 gprepacked, (0-70% ethyl acetate/hexanes) to afford3-[2-deoxy-2-fluoro-3,5-bis-O-(phenylcarbonyl)-3-D-arabinofuranosyl]-7-nitro-3H-imidazo[4,5-b]pyridine.MS: 507 (M+H)⁺.

Step 2:3-[2-deoxy-2-fluoro-3,5-bis-O-(phenylcarbonyl)-β-D-arabinofuranosyl]-3H-imidazo[4,5-b]pyridin-7-amine

To a stirred solution of3-[2-deoxy-2-fluoro-3,5-bis-O-(phenylcarbonyl)-β-D-arabinofuranosyl]-7-nitro-3H-imidazo[4,5-b]pyridine(1380 mg, 2.72 mmol) in methanol (55 mL) at RT was added platinum(IV)oxide (61.9 mg, 0.272 mmol). The reaction mixture was placed under anatmosphere of hydrogen and stirred at RT for 72 h. The catalyst wasremoved by filtration through a plug of CELITE. The filtrate wasconcentrated under reduced pressure, and the resulting residue waspurified by column chromatography on silica gel; 80 g prepacked, ((0-40%(3:1, ethyl acetate:ethanol)/hexanes) to afford3-[2-deoxy-2-fluoro-3,5-bis-O-(phenylcarbonyl)-β-D-arabinofuranosyl]-3H-imidazo[4,5-b]pyridin-7-amine.MS: 477 (M+H)⁺.

Step 3:3-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-3H-imidazo[4,5-b]pyridin-7-amine

To a stirred solution of3-[2-deoxy-2-fluoro-3,5-bis-O-(phenylcarbonyl)-β-D-arabinofuranosyl]-3H-imidazo[4,5-b]pyridin-7-amine(995 mg, 2.09 mmol) in methanol (36 mL) at ambient temperature was addedammonia (7N in methanol, 12 mL, 84.0 mmol). The resulting solution wasbrought to 80° C. and stirred for 5 h. The reaction mixture was cooledto RT and concentrated under reduced pressure. The resulting residue wassuspended in methanol/dichloromethane and sonicated until a solidprecipitated out of solution. Solid was collected by filtering through aglass frit affording3-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-3H-imidazo[4,5-b]pyridin-7-amine.MS: 269 (M+H)⁺.

Step 4:3-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-N-(phenylcarbonyl)-3H-imidazo[4,5-b]pyridin-7-amine

To a stirred solution of3-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-3H-imidazo[4,5-b]pyridin-7-amine(550 mg, 2.05 mmol) in pyridine (6.5 mL) at RT was added TMSCl (2.62 mL,20.5 mmol). The resulting solution was stirred for 1.5 h followed byaddition of benzyol chloride (0.357 mL, 3.08 mmol). After stirring foran additional hour, water (2.15 mL) was added to the reaction mixture,which was then stirred for 45 min. The reaction mixture was cooled to 0°C. and aqueous ammonia (28% w/w) (0.370 mL, 4.79 mmol) was added. Thereaction mixture was returned to RT and stirred for 45 min and thenconcentrated under reduced pressure. The resulting residue was taken upin water (20 mL) and extracted with ethyl acetate (3×40 mL). The organicextracts were combined washed with brine, dried over sodium sulphate andconcentrated under reduced pressure. The resulting residue was purifiedby column chromatography on silica gel; 120 g prepacked, (0-7%methanol/dichloromethane) to afford3-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-N-(phenylcarbonyl)-3H-imidazo[4,5-b]pyridin-7-amine.MS: 373 (M+H)⁺.

Step 5:3-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-2-fluoro-β-D-arabinofuranosyl}-N-(phenylcarbonyl)-3H-imidazo[4,5-b]pyridin-7-amine

To a stirred mixture of3-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-N-(phenylcarbonyl)-3H-imidazo[4,5-b]pyridin-7-amine(185 mg, 0.497 mmol) and 4 Å molecular sieves in pyridine (3 mL) at 0°C. was added 4,4′-dimethoxytrityl chloride (253 mg, 0.745 mmol) in asingle portion. The reaction mixture was allowed to warm to RT and wasstirred for 18 h. Sieves were removed by filtration and the filtrate wasconcentrated under reduced pressure. The resulting residue was taken upin a mixture of methanol/ether and added to water. The phases wereseparated, and the aqueous layer was extracted with ether (3 times). Theorganic extracts were combined, washed with brine, dried over sodiumsulphate and concentrated under reduced pressure. The resulting residuewas purified by column chromatography on silica gel; 40 g prepacked,((0-40% (3:1, ethyl acetate:ethanol)/hexanes) to afford3-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-2-fluoro-β-D-arabinofuranosyl}-N-(phenylcarbonyl)-3H-imidazo[4,5-b]pyridin-7-amine.MS: 675 (M+H)⁺.

Preparation 8:1-((2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-1,5-dihydro-4H-imidazo[4,5-c]pyridin-4-one

Step 1:((2R,3S,4S)-4-acetoxy-3-fluoro-5-(4-oxo-4,5-dihydro-1H-imidazo[4,5-c]pyridin-1-yl)tetrahydrofuran-2-yl)methylbenzoate

To a suspension of 4-(benzyloxy)-1H-imidazo[4,5-c]pyridine (0.795 g,3.53 mmol) and(3S,4S,5R)-5-((benzoyloxy)methyl)-4-fluorotetrahydrofuran-2,3-diyldiacetate (1 g, 2.94 mmol) in ACN (20 mL) and CH₂Cl₂ (10 mL) at 0° C.under Ar was added 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine(1.34 g, 8.815 mmol). The resulting mixture was stirred at 0° C. for 30min. Then, trimethylsilyl trifluoromethanesulfonate (3.92 g, 17.65 mmol)was added to the solution, and it was stirred at 0° C. for 30 min. Then,it was heated to 80° C. for 16 h. The reaction was cooled to rt and sataq NaHCO₃ (10 mL) and water (30 mL) were added. It was extracted withEtOAc (3×50 mL). The combined organic layers was washed with brine,dried over (Na₂SO₄), concentrated, and the residue was purified bysilica gel column chromatography, eluted with 1 to 10% MeOH in CH₂Cl₂ togive the product. LCMS (ES, m/z): 416.3 [M+H]⁺.

Step 2:1-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,5-dihydro-4H-imidazo[4,5-c]pyridin-4-one

To a solution of((2R,3S,4S)-4-acetoxy-3-fluoro-5-(4-oxo-4,5-dihydro-1H-imidazo[4,5-c]pyridin-1-yl)tetrahydrofuran-2-yl)methylbenzoate (2.5 g, 5.3 mmol) in MeOH (10 mL) was added sodium methanolate(3.47 g, 21.2 mmol). The solution was stirred at rt for 1 h. It wasneutralized with AcOH, and the solution was concentrated. The residuewas purified by reverse phase (AQ C18) chromatography eluted with 0 to30% ACN in aq NH₄HCO₃ (5 mM) to give the product. LCMS (ES, m/z): 270.0[M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 11.31 (s, 1H), 8.06 (s, 1H), 7.23(d, J=7.1 Hz, 1H), 6.62 (d, J=7.1 Hz, 1H), 6.37 (d, J=2.9 Hz, 1H), 5.85(d, J=2.8 Hz, 1H), 5.22-4.98 (m, 2H), 4.54 (d, J=17.7 Hz, 1H), 4.28(dtd, J=29.6, 6.0, 3.1 Hz, 1H), 3.93-3.62 (m, 2H).

Step 3:1-((2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-1,5-dihydro-4H-imidazo[4,5-c]pyridin-4-one

To a stirred solution of1-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1H-imidazo[4,5-c]pyridin-4(5H)-one (340 mg, 1.26mmol) in pyridine (3 mL) at rt was added4,4′-(chloro(phenyl)methylene)bis(methoxybenzene) (426 mg, 1.1 mmol).

It was stirred for 4 h. The mixture was concentrated under reducedpressure, and the residue was purified by silica gel columnchromatography eluted with 1 to 10% MeOH in CH₂Cl₂ (0.5% Et₃N) to givethe product. LCMS (ES, m/z): 572.3 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ11.32 (d, J=5.9 Hz, 1H), 7.95 (s, 1H), 7.41 (d, J=7.8 Hz, 2H), 7.37-7.15(m, 8H), 6.86 (dd, J=10.5, 8.6 Hz, 4H), 6.60 (d, J=7.1 Hz, 1H),6.50-6.36 (m, 1H), 5.92 (d, J=2.6 Hz, 1H), 5.77 (s, 1H), 5.27-5.06 (m,1H), 4.65-4.42 (m, 2H), 3.73 (d, J=2.6 Hz, 6H), 3.30-3.24 (m, 1H).

Preparation 9:N-(7-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)imidazo[2,1-f][1,2,4]triazin-4-yl)benzamide

Step 1:(3R,4R,5R)-2-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol

To a stirring mixture of 7-bromoimidazo[2,1-f][1,2,4]triazin-4-amine (41g, 0.19 mol) in THF (0.50 L) at 0° C. was added MeMgBr (3.0M in THF, 66mL, 0.19 mol) dropwise to maintain the internal temperature below 10° C.Bis(chlorodimethylsilyl)ethane (41 g, 190 mmol) was added in oneportion. MeMgBr (3.0M in diethyl ether, 66 mL, 0.19 mol) was then addeddropwise to maintain the internal temperature below 10° C. i-PrMgCl—LiCl(1.3 M in THF, 0.16 L, 0.21 mol) was added while maintaining theinternal temperature below 10° C. A mixture of(3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)dihydrofuran-2(3H)-one(160 g, 0.38 mol) in THF was added dropwise at 0° C., and the mixturewas then allowed to warm to rt and was stirred for 12 h. The mixture wasdiluted with saturated aqueous ammonium chloride (100 mL) and extractedwith ethyl acetate (3×1000 mL). The combined organic layers were driedover sodium sulfate and concentrated under reduced pressure. Theresulting residue was purified by column chromatography (column height:2500 mm, diameter: 1000 mm, 25% to 75% ethyl acetate gradient inhexanes) to afford(3R,4R,5R)-2-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)-tetrahydrofuran-2-ol.

Step 2:7-((3S,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-yl)imidazo[2,1-f][1,2,4]triazin-4-amine

To a stirring mixture of(3R,4R,5R)-2-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol(64 g, 0.12 mmol) in DCM (1.3 L) at 0° C. was added triethylsilane (81g, 0.69 mol), and then boron trifluoride diethyl etherate (21 g, 0.15mol). The mixture was then allowed to warm to 25° C., and the mixturewas stirred for 1 h. More boron trifluoride diethyl etherate (57 g, 0.40mol) was added, and the mixture was then heated to 35° C. for 4 h. Uponcooling to rt, the mixture was quenched with saturated aqueous sodiumbicarbonate (200 mL) and then extracted with ethyl acetate (3×300 mL).The combined organic layers were dried over sodium sulfate andconcentrated under reduced pressure. The resulting residue was purifiedby column chromatography (15-75% ethyl acetate gradient in hexanes) toafford7-((3S,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-yl)imidazo[2,1-f][1,2,4]triazin-4-amine.MS (ES, m/z)=538 [M+H]+.

Step 3:(3R,4S,5R)-2-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-5-(hydroxymethyl)-tetrahydrofuran-3,4-diol

To a stirring mixture of7-((3S,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)-tetrahydrofuran-2-yl)imidazo[2,1-f][1,2,4]triazin-4-amine(12 g, 22 mmol) in DCM (850 mL) at −78° C. was added boron trichloride(18 g, 0.16 mol) dropwise. Upon completion, the mixture was stirred at−78° C. for 3 h. After 3 h, the mixture was quenched with methanol (50mL) at −78° C., and the mixture was allowed to warm to 25° C. Themixture was concentrated under reduced pressure. The residue waspurified by column chromatography (9-25% methanol gradient indichloromethane) to afford(3R,4S,5R)-2-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol.

Step 4.(6aR,8S,9S,9aS)-8-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-ol

To a stirred mixture of(2S,3R,4S,5R)-2-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol(4.0 g, 15 mmol) in pyridine (0.10 L) was added1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (5.8 mL, 18 mmol). After 3h, the mixture was diluted with toluene (50 mL) and then concentrated.The resulting mixture was taken up in DCM and methanol and then silicagel (40 g) was added. The mixture was concentrated, placed under vacuumfor 1 h and then purified by column chromatography (0-80% ethyl acetategradient in hexanes) to afford(6aR,8S,9S,9aS)-8-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-ol.MS (ES, m/z)=510 [M+H]+.

Step 5:O-((6aR,8S,9S,9aR)-8-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-yl)1H-imidazole-1-carbothioate

To a mixture of(6aR,8S,9S,9aS)-8-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-ol(6.45 g, 12.7 mmol) in acetonitrile (63.0 mL) and pyridine (63.0 mL) wasadded 1,1′-thiocarbonyldiimidazole (2.71 g, 15.2 mmol). After 90 min,more 1,1′-thiocarbonyldiimidazole (2.71 g, 15.2 mmol) was added, and themixture was stirred overnight. After stirring overnight, the mixture wasconcentrated and purified by column chromatography (0-100% ethyl acetategradient in hexanes) to affordO-((6aR,8S,9S,9aR)-8-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-yl)1H-imidazole-1-carbothioate. MS (ES, m/z)=620 [M+H]+.

Step 6:7-((6aR,8R,9aS)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)imidazo[2,1-f][1,2,4]triazin-4-amine

To a mixture ofO-((6aR,8S,9S,9aR)-8-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-yl)(5.65 g, 9.11 mmol) in toluene (91.0 mL) was added2,2′-azobis(2-methylpropionitrile (0.300 g, 1.82 mmol) andtris(trimethylsilyl)silane (4.22 mL, 13.7 mmol). The mixture was heatedto 85° C. for 30 min. After 30 min, the mixture was allowed to cool tort and placed directly on the column and purified (0-80% ethyl acetategradient in hexanes) to afford7-((6aR,8R,9aS)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)imidazo[2,1-f][1,2,4]triazin-4-amine.MS (ES, m/z)=494 [M+H]+ 494.

Step 7:N-benzoyl-N-(7-((6aR,8R,9aS)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)imidazo[2,1-f][1,2,4]triazin-4-yl)benzamide

To a mixture of7-((6aR,8R,9aS)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)imidazo[2,1-f][1,2,4]triazin-4-amine(15.7 g, 31.8 mmol) in pyridine (64.0 mL) was added benzoyl chloride(11.0 mL, 95.0 mmol), and the mixture was heated to 50° C. for 45 min.After 45 min, the mixture was allowed to cool to rt. After cooling, aprecipitate formed and was filtered off. The filtrate was diluted withDCM (50 mL) and toluene (50 mL). The mixture was concentrated to about50 mL. The mixture was filtered, and the solids washed with DCM. Thefiltrate and washes were combined, loaded onto a column, and purified(0-50% ethyl acetate gradient in hexanes) to affordN-benzoyl-N-(7-((6aR,8R,9aS)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)imidazo[2,1-f][1,2,4]triazin-4-yl)benzamide.MS (ES, m/z)=702 [M+H]+.

Step 8:N-(7-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)imidazo[2,1-f][1,2,4]triazin-4-yl)benzamide

To a mixture ofN-benzoyl-N-(7-((6aR,8R,9aS)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)imidazo[2,1-f][1,2,4]triazin-4-yl)benzamide(10 g, 14 mmol) in tetrahydrofuran (0.14 L) was added TBAF ((1.0M inTHF, 29 mL, 29 mmol), and the mixture was stirred for 1 h. After 1 h,the mixture was concentrated and purified by column chromatography toaffordN-(7-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydro-furan-2-yl)imidazo[2,1-f][1,2,4]triazin-4-yl)benzamide.MS (ES, m/z)=356 [M+H]+.

Step 9:N-(7-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)imidazo[2,1-f][1,2,4]triazin-4-yl)benzamide

To a mixture ofN-(7-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)imidazo[2,1-f][1,2,4]triazin-4-yl)benzamide(6.1 g, 17 mmol) in pyridine (86 mL) at 0° C. was added4,4′-(chloro(phenyl)methylene)bis(methoxybenzene) (5.8 g, 17 mmol), andthe mixture was allowed to warm to RT overnight. After stirringovernight, the mixture was diluted with toluene and then concentratedunder reduced pressure to afford the crude product. The crude productwas purified by silica gel chromatography (0-100% ethyl acetate gradientin hexanes) to affordN-(7-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)imidazo[2,1-f][1,2,4]triazin-4-yl)benzamide.MS (ES, m/z)=658 [M+H]+.

Preparation 10:(2R,3R,4S,5R)-2-(4-amino-1H-[1,2,3]triazolo[4,5-c]pyridin-1-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol

Step 1:(2R,3R,4R,5R)-2-(acetoxymethyl)-5-(4-chloro-1H-[1,2,3]triazolo[4,5-c]pyridin-1-yl)tetrahydrofuran-3,4-diyldiacetate

To a suspension of 4-chloro-1H-[1,2,3]triazolo[4,5-c]pyridine (1.0 g,6.5 mmol) and (3R,4R,5R)-5-(acetoxymethyl)tetrahydrofuran-2,3,4-triyltriacetate (3.1 g, 9.7 mmol) in nitromethane (50 mL) was added BF₃.Et₂O(1.23 mL, 9.7 mmol), and the resulting mixture was heated at 50° C. for2 h. The reaction mixture was cooled to rt, diluted with 100 mL of DCMand washed with sat aq. NaHCO₃ (100 mL) and brine (100 mL). Theseparated organic layer was dried over Na₂SO₄ and concentrated in vacuo.The residue was purified by a silica gel column, eluting with 0-100%EtOAc:EtOH (3:1)/Hexane. LCMS (ES, m/z): 413.07 [M+H]⁺. ¹H NMR (500 MHz,Chloroform-d) δ 8.40 (d, J=5.8 Hz, 1H), 7.56 (d, J=5.8 Hz, 1H), 6.43 (d,J=4.1 Hz, 1H), 6.17 (dd, J=5.3, 4.1 Hz, 1H), 5.74 (t, J=5.3 Hz, 1H),5.32 (s, 1H), 4.58 (ddd, J=5.3, 3.9, 2.9 Hz, 1H), 4.42 (dd, J=12.5, 3.0Hz, 1H), 4.25 (dd, J=12.5, 3.9 Hz, 1H), 2.17 (d, J=18.8 Hz, 6H), 2.03(s, 3H).

Step 2:(2R,3R,4S,5R)-2-(4-amino-1H-[1,2,3]triazolo[4,5-c]pyridin-1-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol

To a solution of(2R,3R,4R,5R)-2-(acetoxymethyl)-5-(4-chloro-1H-[1,2,3]triazolo[4,5-c]pyridin-1-yl)tetrahydrofuran-3,4-diyldiacetate (1.2 g, 2.9 mmol) in MeOH (8.3 mL) was added a 7N solution ofammonia in MeOH (8.3 mmol, 58.1 mmol). The reaction mixture was stirredat 150° C. in a sealed Q-Tube™ Pressure Tube Reactor for 18 h. Excesssolvent was removed under reduced pressure, and the residue was purifiedby a reverse phase silica gel column, eluting with 0-10%Acetonitrile/H₂O containing 0.05% TFA. LCMS (ES, m/z): 268.17 [M+H]⁺. ¹HNMR (500 MHz, DMSO-d₆) δ 13.87 (s, 2H), 9.34 (s, 4H), 7.89 (d, J=7.1 Hz,2H), 7.51 (d, J=7.0 Hz, 2H), 7.42 (s, 4H), 7.32 (s, 6H), 7.22 (s, 5H),6.69 (s, 2H), 6.30 (d, J=4.9 Hz, 2H), 5.72 (s, 2H), 5.41 (s, 2H),4.75-4.67 (m, 0H), 4.65 (t, J=5.0 Hz, 2H), 4.27 (t, J=4.6 Hz, 2H), 4.07(q, J=4.2 Hz, 2H), 3.86-3.67 (m, 2H), 3.69-3.58 (m, 2H), 3.54 (dd,J=12.0, 4.7 Hz, 2H), 3.48-3.38 (m, 1H), 3.28 (s, 1H), 3.23 (s, 0H), 1.76(d, J=0.5 Hz, 5H), 1.11 (dt, J=26.0, 7.1 Hz, 1H).

Preparation 11:9-{3-azido-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-deoxy-β-D-ribofuranosyl}-2-[(2-methylpropanoyl)amino]-1,9-dihydro-6H-purin-6-one

The title compound was prepared according to published procedures(Nucleosides, Nucleotides & Nucleic Acids 2005, 24(10-12), 1707-1727).

Preparation 12:9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-deoxy-3-fluoro-β-D-xylofuranosyl}-2-[(2-methylpropanoyl)amino]-1,9-dihydro-6H-purin-6-one

The title compound was prepared according to published procedures(Tetrahedron Letters, 1989, 30(24), 3171-3174).

Preparation 13:1-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-deoxy-3-fluoro-β-D-ribofuranosyl}-6-[(2-methylpropanoyl)amino]-1,5-dihydro-4H-imidazo[4,5-c]pyridin-4-one

The title compound was prepared according to published procedures(WO2002057425).

Preparation 14:N-(3-((2R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,3-difluoro-4-hydroxytetrahydrofuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)benzamide

Step 1:(((2R,3R)-3-(benzoyloxy)-5-((diphenoxyphosphoryl)oxy)-4,4-difluorotetrahydrofuran-2-yl)methylbenzoate

To((2R,3R)-3-(benzoyloxy)-4,4-difluoro-5-hydroxytetrahydrofuran-2-yl)methylbenzoate (20.0 g, 52.9 mmol) in toluene (150 mL) at 0° C. were addedEt₃N (7.74 mL, 55.5 mmol) and diphenyl phosphoryl chloride (12.1 mL,58.2 mmol) in toluene (20 mL) dropwise. The reaction was warmed to rtand stirred for 3 h. LCMS showed completion of the reaction (m/e=611).Water (30 mL) and aq HCl (1 M, 5 mL) were added. Layers were separated,and the aq layer was extracted with CH₂Cl₂ (2×30 mL). The combinedorganic solution was washed with sat aq NaHCO₃ (20 mL), and Brine (20mL), dried (MgSO₄), and concentrated. It was purified by silica columnchromatography eluting with 0 to 30% EtOAc in Hex to give the product.LCMS (ES, m/z): 611.3 [M+H]⁺.

Step 2:((2R,3R,5R)-3-(benzoyloxy)-5-bromo-4,4-difluorotetrahydrofuran-2-yl)methylbenzoate

To((2R,3R)-3-(benzoyloxy)-5-((diphenoxyphosphoryl)oxy)-4,4-difluorotetrahydrofuran-2-yl)methylbenzoate (23.8 g, 39.0 mmol) at 0° C. was added HBr in acetic acid (33%,51.2 mL, 292 mmol). It was stirred as it warmed to rt. After 6 h, LCMSshowed completion of the reaction (m/e=441 and 443). CH₂Cl₂ (200 mL) wasadded, and the organic layer was washed with water (2×50 mL), sat aqNaHCO₃ (2×50 mL) and brine (50 mL). It was dried (MgSO₄) andconcentrated to give the crude product. It was used in the next stepwithout purification. LCMS (ES, m/z): 441.1, 443.1 [M+H]⁺.

Step 3:(2R,3R,5R)-5-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((benzoyloxy)methyl)-4,4-difluorotetrahydrofuran-3-ylbenzoate

To a mixture of 3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-amine (3.21 g, 23.6mmol) in NMP (60 mL) was added NaH (60%, 0.979 g, 24.5 mmol). Themixture was vigorously stirred, and after 1 h, generation of bubbles hadcompletely ceased. To the mixture was added to((2R,3R,5R)-3-(benzoyloxy)-5-bromo-4,4-difluorotetrahydrofuran-2-yl)methylbenzoate (neat, 8.00 g, 18.1 mmol). The mixture was stirred vigorouslyfor 30 min. Then, it was heated to 90° C. for 5 h. It was cooled to rt,and CH₂Cl₂ (300 mL) and water (150 mL) were added. The phases wereseparated, and the organic layer was washed with water (8×150 mL) andbrine (50 mL), dried (MgSO₄) and concentrated. The residue was purifiedby silica column chromatography eluting with 0% to 30% EtOAc to give thedesired product. LCMS (ES, m/z): 497.1 [M+H]⁺. ¹H NMR (500 MHz,Chloroform-d) δ 8.44 (s, 1H), 8.15-8.08 (m, 2H), 8.08-7.99 (m, 2H), 7.63(ddt, J=8.7, 7.1, 1.3 Hz, 1H), 7.56-7.44 (m, 3H), 7.40-7.32 (m, 2H),6.79-6.68 (m, 2H), 6.04 (bs, 2H), 4.92-4.84 (m, 1H), 4.80-4.72 (m, 2H).

Step 4.(2R,3R,5R)-5-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-4,4-difluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol

To(2R,3R,5R)-5-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((benzoyloxy)methyl)-4,4-difluorotetrahydrofuran-3-yl benzoate (1.05 g, 2.11 mmol)was added ammonia in MeOH (7N, 9.0 mL, 63 mmol), and the mixture wasstirred for 24 h. LCMS showed completion of reaction (m/e=289). It wasconcentrated and purified by silica column chromatography eluting with 0to 15% MeOH in CH₂Cl₂ to give the desired product. LCMS (ES, m/z): 289.1[M+H]⁺. ¹H-NMR (500 MHz, DMSO-d₆): δ 8.65 (s, 1H), 8.36 (s, 1H), 8.32(s, 1H), 6.65 (d, J=12.4 Hz, 1H), 6.40 (d, J=6.7 Hz, 1H), 5.03 (dd,J=6.4, 5.3 Hz, 1H), 4.83 (dq, J=16.5, 8.9 Hz, 1H), 4.00 (t, J=6.7 Hz,1H), 3.77 (ddd, J=12.3, 5.2, 2.5 Hz, 1H), 3.68 (dt, J=12.5, 6.3 Hz, 1H).

Step 5.N-(3-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)benzamide

To a solution of(2R,3R,5R)-5-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-4,4-difluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol(0.57 g, 2.0 mmol) in pyridine (15 mL) at 0° C. was added TMSCl (0.55mL, 4.3 mmol). It was warmed to rt and stirred for 1 h. Then, it wasrecooled to 0° C. and BzCl (0.34 mL, 2.9 mmol) was added dropwise. Thereaction was slowly warmed rt over 2 h. LCMS showed completion ofreaction (m/e=393, 497). It was cooled to 0° C., and ammonium hydroxide(28%, 1.1 mL, 7.9 mmol) was added. After 30 min, it was concentrated andpurified by silica column chromatography eluting with 0 to 10% MeOH inCH₂Cl₂ to give the product. LCMS (ES, m/z): 393.3 [M+H]⁺. ¹H-NMR (500MHz, DMSO-d₆): δ 12.02 (s, 1H), 9.01 (s, 1H), 8.13-8.07 (m, 2H),7.73-7.66 (m, 1H), 7.64-7.55 (m, 2H), 6.86 (d, J=11.6 Hz, 1H), 6.46 (d,J=6.7 Hz, 1H), 5.01 (dd, J=6.3, 5.4 Hz, 1H), 4.86 (dt, J=16.7, 8.3 Hz,1H), 4.04 (ddd, J=8.8, 6.2, 2.6 Hz, 1H), 3.78 (ddd, J=12.3, 5.4, 2.7 Hz,1H), 3.68 (dt, J=12.4, 6.2 Hz, 1H).

Step 6.N-(3-((2R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,3-difluoro-4-hydroxytetrahydrofuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)benzamide

To a solution ofN-(3-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)benzamide(0.68 g, 1.7 mmol) in pyridine (15 mL) at 0° C. was added DMTrCl (0.65g, 1.9 mmol). It was stirred at rt for 1 h. LCMS showed a peak with thedesired mass (m/e=695). It was partly concentrated (to 5 mL), and EtOAc(20 mL) and water (10 mL) were added. The phases were separated, and theaqueous layer was extracted with EtOAc (2×10 mL). The combined organicportions were washed with brine, dried (MgSO₄), concentrated andpurified by silica column chromatography eluting with 0% to 60% EtOAc inHex to give the product. LCMS (ES, m/z): 695.2 [M+H]⁺. ¹H-NMR (500 MHz,DMSO-d₆): δ 12.05 (s, 1H), 8.93 (s, 1H), 8.11 (d, J=7.6 Hz, 2H), 7.70(m, 1H), 7.60 (t, J=7.7 Hz, 2H), 7.38-7.32 (m, 2H), 7.25-7.13 (m, 7H),6.96 (d, J=11.9 Hz, 1H), 6.84-6.73 (m, 4H), 6.48 (d, J=6.9 Hz, 1H), 4.97(dq, J=16.7, 8.3 Hz, 1H), 4.23 (m, 1H), 3.70 (s, 3H), 3.69 (s, 3H),3.42-3.36 (m, 1H), 3.32-3.28 (m, 1H).

Preparation 15:N-(7-((2R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,3-difluoro-4-hydroxytetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide

Step 1.((2R,3R,5R)-3-(benzoyloxy)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4,4-difluorotetrahydrofuran-2-yl)methylbenzoate

To a solution of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (4.06 g, 26.4mmol) and((2R,3R)-3-(benzoyloxy)-4,4-difluoro-5-hydroxytetrahydrofuran-2-yl)methylbenzoate (10.0 g, 26.4 mmol) and triphenylphosphine (20.80 g, 79 mmol)in THF (100 mL) was added(E)-diazene-1,2-diylbis(piperidin-1-ylmethanone) (20.01 g, 79 mmol)dropwise. It was slowly warmed to rt. After stirring for 2 h, themixture was concentrated. The residue was purified by silica gel columnchromatography using 0-20% EtOAc in Petroleum Ether to give the product.LCMS (ES, m/z): 514.3 [M+H]⁺. ¹H-NMR (300 MHz, DMSO-d₆): δ 8.71 (s, 1H),8.08-7.87 (m, 5H), 7.76-7.42 (m, 6H), 6.98 (dd, J=10.3, 7.9 Hz, 1H),6.84 (d, J=3.8 Hz, 1H), 6.29 (ddd, J=10.0, 6.0, 3.8 Hz, 1H), 5.44 (q,J=5.5 Hz, 1H), 4.79-4.60 (m, 2H).

Step 2.(2R,3R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4,4-difluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol

A solution of((2R,3R,5R)-3-(benzoyloxy)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4,4-difluorotetrahydrofuran-2-yl)methylbenzoate (1.9 g, 3.70 mmol) in NH₃/2-propanol (saturated at −78° C., 100mL) was stirred at 90° C. for 16 h. It was cooled to rt, concentrated,and purified by silica gel column chromatography using 0-10% MeOH inCH₂Cl₂ to give the product. LCMS (ES, m/z): 287.1 [M+H]⁺. ¹H-NMR (300MHz, DMSO-d₆): δ 8.06 (s, 1H), 7.40-7.25 (m, 1H), 7.14 (s, 2H), 6.82 (d,J=5.9 Hz, 1H), 6.60 (d, J=3.7 Hz, 1H), 6.34 (dd, J=16.2, 2.1 Hz, 1H),4.88 (t, J=5.7 Hz, 1H), 4.27 (ddd, J=9.9, 6.2, 3.6 Hz, 1H), 4.08 (dq,J=6.8, 4.1, 3.5 Hz, 1H), 3.78 (dt, J=11.1, 5.4 Hz, 1H), 3.62 (dt,J=11.4, 5.8 Hz, 1H).

Step 3:N-(7-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-hydroxymethyl)tetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide

To a solution of(2R,3R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4,4-difluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol(900 mg, 3.14 mmol) in pyridine (15 mL) at 0° C. was addedchlorotrimethylsilane (3.42 g, 31.4 mmol). It was warmed to rt over 1 hand benzoyl chloride (663 mg, 4.72 mmol) was added dropwise. After 2 h,NH₄OH (28%, 15.00 mL) was added, and it was stirred for 0.5 h. Theresulting mixture was concentrated, and the residue was purified bysilica gel column chromatography using 0-10% MeOH in CH₂Cl₂ to give theproduct. LCMS (ES, m/z): 391.1 [M+H]⁺. ¹H-NMR (300 MHz, DMSO-d₆): δ 8.61(s, 1H), 8.08-7.98 (m, 2H), 7.97-7.91 (m, 1H), 7.67-7.51 (m, 2H),7.50-7.47 (m, 1H), 6.70 (d, J=3.9 Hz, 1H), 6.56 (dd, J=15.6, 1.5 Hz,1H), 4.32 (dd, J=9.4, 3.6 Hz, 1H), 4.16 (d, J=5.6 Hz, 1H), 3.80 (dd,J=11.5, 5.2 Hz, 1H), 3.66 (dd, J=11.5, 6.5 Hz, 1H), 3.12 (s, 2H).

Step 4.N-(7-((2R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,3-difluoro-4-hydroxytetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide

To a solution ofN-(7-((2S,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide(1.1 g, 2.82 mmol) in pyridine (12 mL) at 0° C. was added4,4′-(chloro(phenyl)methylene)-bis(methoxybenzene) (0.955 g, 2.82 mmol).It was warmed to RT and stirred for 3 h. The mixture was concentrated.The product was purified by silica gel column chromatography using 0-10%MeOH in CH₂Cl₂ to give the product. LCMS (ES, m/z): 693.3 [M+H]⁺. ¹H-NMR(300 MHz, DMSO-d₆): δ 11.25 (s, 1H), 8.67 (s, 1H), 8.09-8.01 (m, 2H),7.60-7.48 (m, 2H), 7.47-7.36 (m, 3H), 7.33-7.18 (m, 7H), 6.91-6.78 (m,4H), 6.75-6.57 (m, 3H), 5.76 (s, 1H), 4.55-4.33 (m, 2H), 3.74 (s, 6H),3.44 (t, J=8.7 Hz, 1H), 3.35 (s, 1H).

Preparation 16:N-(9-((2R,3S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

Step 1:(2R,4S)-4-(benzyloxy)-4-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-3,3-difluoro-2-hydroxybutylbenzoate

To a solution of(2R,4S)-4-(benzyloxy)-4-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-3,3-difluorobutane-1,2-diol(3.50 g, 10.5 mmol) in CH₂Cl₂ (52 mL) and pyridine (26 mL) at −70° C.was added benzoyl chloride (1.48 g, 10.5 mmol) in CH₂Cl₂ (11 mL)dropwise over 50 min. After 2 h, methanol (150 mL) was added. Themixture was stirred at RT for 0.5 h. Water (200 mL) was added. Layerswere separated, and the aq layer was extracted with ether (4×150 mL).The combined organic layers were washed with aq HCl (1 N, 2×150 mL), sataq NaHCO₃ (2×150 mL) and brine (2×150 mL). It was dried (Na₂SO₄),concentrated and purified by silica gel column chromatography(EtOAc/petroleum ether=1/10) to give the product. ¹H-NMR (400 MHz,CDCl₃): δ 8.11-8.01 (m, 2H), 7.61-7.55 (m, 1H), 7.48-7.31 (m, 7H),4.89-4.53 (m, 4H), 4.46-4.41 (m, 1H), 4.40-4.29 (m, 1H), 4.17-4.02 (m,2H), 3.98-3.84 (m, 0.5H), 3.74-3.66 (m, 0.5H), 1.46 (s, 3H), 1.28 (s,3H). ¹⁹F-NMR: (376 MHz, CDCl₃): δ −106.8 (d, J=270.7 Hz, 1F), −119.2 (d,J=270.7 Hz, 1F).

Step 2:((2R,4S)-4-(benzyloxy)-3,3-difluoro-5-hydroxytetrahydrofuran-2-yl)methylbenzoate

(2R,4S)-4-(benzyloxy)-4-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-3,3-difluoro-2-hydroxybutylbenzoate (3.00 g, 6.87 mmol) was dissolved in aq AcOH (75%, 66 mL). Itwas stirred at 50° C. for 3 h. It was partly concentrated. The residuewas redissolved in acetone (33 mL). To the solution at rt was addedsodium periodate (1.20 g, 5.61 mmol) in water (33 mL). After 1.5 h, thesolid formed was filtered off and washed with acetone. The filtrate wasconcentrated. Water and CH₂Cl₂ were added, and layers were separated.The aq layer was extracted with CH₂Cl₂ (4×150 mL). The combined organiclayers were dried (Na₂SO₄), concentrated, and purified by silica gelcolumn chromatography (ethyl acetate/petroleum ether=1/5) to give theproduct. ¹H-NMR (400 MHz, CDCl₃): δ 8.05-8.01 (m, 2H), 7.59-7.52 (m,1H), 7.46-7.35 (m, 7H), 5.49-5.42 (m, 1H), 4.99-4.72 (m, 1H), 4.67-4.47(m, 4H), 4.11-3.80 (m, 1H). ¹⁹F-NMR: (376 MHz, CDCl₃): δ −117.1 (d,J=240.6 Hz, 1F), −117.9 (d, J=251.9 Hz, 1F).

Step 3:((2R,4S)-5-acetoxy-4-(benzyloxy)-3,3-difluorotetrahydrofuran-2-yl)methylbenzoate

To a solution of((2R,4S)-4-(benzyloxy)-3,3-difluoro-5-hydroxytetrahydrofuran-2-yl)methylbenzoate (2.40 g, 6.59 mmol) in CH₂Cl₂ (66 mL) at rt was addedN,N-dimethylpyridin-4-amine (0.080 g, 0.659 mmol) and acetic anhydride(4.03 g, 39.5 mmol) dropwise. After 6 h, it was quenched by addition ofsat aq NaHCO₃ (30 mL). Layers were separated, and the aq layer wasextracted with CH₂Cl₂ (3×150 mL). The combined organic layers werewashed with water (2×150 mL) and brine (2×100 mL), dried (Na₂SO₄),concentrated and purified by silica gel column chromatography (ethylacetate/petroleum ether=1/7) to give the product. ¹H-NMR (300 MHz,CDCl₃): δ 8.08-7.89 (m, 2H), 7.61-7.48 (m, 1H), 7.47-7.25 (m, 7H), 6.15(d, J=6.0 Hz, 1H), 4.89-4.75 (m, 1H), 4.73-4.40 (m, 4H), 4.18-4.02 (m,1H), 1.98 (s, 3H). ¹⁹F-NMR: (282 MHz, CDCl₃): δ −116.5 (d, J=248.2 Hz,1F), −120.9 (d, J=248.2 Hz, 1F).

Step 4:((2R,4S)-5-acetoxy-3,3-difluoro-4-hydroxytetrahydrofuran-2-yl)methylbenzoate

To a solution of((2R,4S)-5-acetoxy-4-(benzyloxy)-3,3-difluorotetrahydrofuran-2-yl)methylbenzoate (2.50 g, 6.15 mmol) in EtOAc (60 mL) was added sodium bromate(5.57 g, 36.9 mmol) in water (46 mL). The mixture was stirredvigorously, and to it was added sodium dithionite (6.43 g, 36.9 mmol) inwater (92 mL) dropwise over 1 h. After 5 h, layers were separated, andthe aq layer was extracted with EtOAc (5×150 mL). The combined organiclayers were washed with sat aq Na₂S₂O₃ (2×150 mL) and brine (2×150 mL),dried (Na₂SO₄), concentrated, and purified by silica gel columnchromatography (ethyl acetate/petroleum ether=1/5) to give the product.¹H-NMR (300 MHz, CDCl₃): δ 8.10-7.89 (m, 2H), 7.63-7.50 (m, 1H),7.48-7.30 (m, 2H), 6.09 (d, J=6.0 Hz, 1H), 4.71-4.42 (m, 3H), 4.36-4.26(m, 1H), 2.04 (s, 3H). 19F-NMR: (282 MHz, CDCl₃): δ −119.5 (d, J=248.2Hz, 1F), −122.0 (d, J=248.2 Hz, 1F).

Step 5:(3S,5R)-5-((benzoyloxy)methyl)-4,4-difluorotetrahydrofuran-2,3-diyldiacetate

To a solution of((2R,4S)-5-acetoxy-3,3-difluoro-4-hydroxytetrahydrofuran-2-yl)methylbenzoate (2.00 g, 6.32 mmol) in CH₂Cl₂ (84 mL) at rt was added DMAP(0.08 g, 0.632 mmol) and acetic anhydride (3.87 g, 37.9 mmol) dropwise.After 6 h, it was quenched by addition of sat aq NaHCO₃ (30 mL). Layerswere separated, and the aq layer was extracted with CH₂C₁₂ (3×140 mL).The combined organic layers were washed with water (2×140 mL) and brine(2×140 mL), dried (Na₂SO₄), concentrated and purified by silica gelcolumn chromatography (ethyl acetate/petroleum ether=1/6) to give theproduct. ¹H-NMR (400 MHz, CDCl₃): δ 8.11-8.01 (m, 2H), 7.63-7.54 (m,1H), 7.50-7.41 (m, 2H), 6.20 (d, J=4.0 Hz, 1H), 5.39 (d, J=8.0 Hz, 1H),4.69-4.48 (m, 3H), 2.23 (s, 3H), 2.08 (s, 3H). ¹⁹F-NMR: (376 MHz,CDCl₃): δ −117.6 (d, J=251.9 Hz, 1F), −119.5 (d, J=251.9 Hz, 1F).

Step 6:((2R,4S,5R)-4-acetoxy-5-(6-chloro-2-isobutyramido-9H-purin-9-yl)-3,3-difluorotetrahydrofuran-2-yl)methylbenzoate

To a solution of (3S,5R)-5-((benzoyloxy)methyl)-4,4-difluorotetrahydrofuran-2,3-diyldiacetate (2.20 g, 6.14 mmol) andN-(6-chloro-9H-purin-2-yl)isobutyramide (1.77 g, 7.37 mmol) in ACN (80mL) at 0° C. was added 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine(2.80 mL, 18.4 mmol). After 0.5 h, trimethylsilyltrifluoromethanesulfonate (6.82 mL, 36.8 mmol) was added dropwise to thereaction at 0° C. After 0.5 h, it was heated at 80° C. for 16 h. Thereaction was then quenched by the addition of water (150 mL). Layerswere separated, and the aq layer was extracted with EtOAc (3×150 mL).The combined organic layers were washed with sat aq NaHCO₃ (2×150 mL)and brine (2×150 mL), dried (Na₂SO₄), concentrated and purified bysilica gel column chromatography (ethyl acetate/petroleum ether=1/1) togive the product. ¹H-NMR (400 MHz, CDCl₃): δ 8.10 (s, 1H), 8.08-7.98 (m,2H), 7.64-7.53 (m, 1H), 7.51-7.40 (m, 2H), 6.25 (d, J=4.0 Hz, 1H),5.98-5.93 (m, 1H), 4.85-4.53 (m, 3H), 2.92-2.80 (m, 1H), 2.22 (s, 3H)1.28 (d, J=4.0 Hz, 6H). ¹⁹F-NMR: (376 MHz, CDCl₃): δ −116.7 (d, J=248.2Hz, 1F), −118.1 (d, J=248.2 Hz, 1F).

Step 7:((2R,4S,5R)-5-(2-acetamido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-acetoxy-3,3-difluorotetrahydrofuran-2-yl)methylbenzoate

To a solution of((2R,4S,5R)-4-acetoxy-5-(6-chloro-2-isobutyramido-9H-purin-9-yl)-3,3-difluorotetrahydrofuran-2-yl)methylbenzoate (1.80 g, 3.35 mmol) in AcOH (29 mL) was added Sodium acetate(1.37 g, 16.7 mmol) and acetic anhydride (29 mL). The reaction wasstirred at 125° C. for 2.5 h. It was cooled to rt, and MeOH (50 mL) wasadded. The mixture was concentrated under reduced pressure, and theresidue was coevaporated with ethanol (2×50 mL). DCM (150 mL) and water(150 mL) were added, and layers were separated. The organic phase waswashed with sat aq NaHCO₃ (2×150 mL), dried (Na₂SO₄), and concentratedto give the product. LCMS (ES, m/z): 492.1 [M+H]⁺.

Step 8:2-amino-9-((2R,3S,5R)-4,4-difluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one

To((2R,4S,5R)-5-(2-acetamido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-acetoxy-3,3-difluorotetrahydrofuran-2-yl)methylbenzoate (neat, 1.80 g, 3.66 mmol) was added NH₃ in MeOH (7 M, 90 mL,MeOH). It was stirred at rt for 60 h. It was concentrated and purifiedby reverse phase (C18) chromatography eluting with 5-20% MeCN in aqNH₄HCO₃ (5 mM) to give the product. ¹H-NMR (300 MHz, DMSO-d₆): δ 10.7(s, 1H), 7.95 (s, 1H), 6.56-6.44 (m, 3H), 5.62 (d, J=6.0 Hz, 1H), 5.32(t, J=5.4 Hz, 1H), 4.90-4.77 (m, 1H), 4.23-4.08 (m, 1H), 3.68-3.52 (m,2H). ¹⁹F-NMR: (282 MHz, DMSO-d₆): δ −113.1 (d, J=234.1 Hz, 1F), −121.8(d, J=234.1 Hz, 1F).

Step 9.N-(9-((2R,3S,5R)-4,4-difluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

2-amino-9-((2R,3S,5R)-4,4-difluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one(800 mg, 2.64 mmol) was co-evaporated with pyridine (3×3 mL). It wasre-suspended in pyridine (13 mL). To the mixture at 0° C. was addedchlorotrimethylsilane (1.686 mL, 13.19 mmol) dropwise. It was warmed tort and stirred for 2 h. The reaction was cooled to 0° C., and isobutyricanhydride (0.656 mL, 3.96 mmol) was added dropwise. It was warmed to rt,stirred for 2 h, and then water (4 mL) and NH₄OH (8 mL) were added tothe reaction. After 30 min, it was concentrated. The residue waspurified by flash column chromatography with 0-10% MeOH in CH₂Cl₂ togive the product. LCMS (ES, m/z): 374.1 [M+H]⁺. ¹H-NMR: (300 MHz,DMSO-d₆) δ 12.11 (s, 1H), 11.69 (s, 1H), 8.30 (s, 1H), 6.58 (d, J=6.0Hz, 1H), 5.74 (dd, J=9.0, 3.0 Hz, 1H), 5.33 (t, J=6.0 Hz, 1H), 4.96-4.83(m, 1H), 4.26-4.17 (m, 1H), 3.72-3.62 (m, 2H), 2.80-2.71 (m, 1H), 1.11(d, J=9.0 Hz, 6H).

Step 10:N-(9-((2R,3S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

N-(9-((2R,3S,5R)-4,4-difluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide(640 mg, 1.714 mmol) was co-evaporated with pyridine (3×3 mL) and thenre-suspended in pyridine (5.7 mL). To the suspension was added4,4′-(chloro(phenyl)methylene)bis(methoxybenzene) (639 mg, 1.886 mmol),and it was stirred for 16 h. Then, it was concentrated and then,co-evaporated with toluene (3×20 mL). The crude was purified by silicacolumn chromatography eluting with 1 to 30% MeOH in CH₂Cl₂ (containing1% Et₃N) to give the product. LCMS (ES, m/z): 676.3 [M+H]⁺. ¹H-NMR: (300MHz, DMSO-d₆) δ 12.11 (s, 1H), 11.61 (s, 1H), 8.15 (s, 1H), 7.34 (J=6.0,3.0 Hz, 2H), 7.28-7.19 (m, 7H), 6.85-6.80 (m, 4H), 6.71 (d, J=6.0 Hz,1H), 5.78 (d, J=6.0 Hz, 1H), 5.13-5.05 (m, 1H), 4.46-4.39 (m, 1H), 3.71(s, 6H), 3.46-3.40 (m, 1H), 3.22-3.18 (m, 1H), 2.78-2.70 (m, 1H), 1.11(d, J=9.0 Hz, 6H).

Preparation 17:N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-fluoro-3-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

Step 1:2-amino-9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-hydroxytetrahydro-4H-thieno[3,2-d][1,3,2]dioxasilin-6-yl)-1,9-dihydro-6H-purin-6-one

To a stirred suspension of2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-1H-purin-6(9H)-one(15 g, 50.1 mmol) in DMF (150 mL) at 0° C. under Ar was injecteddi-tert-butylsilanediyl bis(trifluoromethanesulfonate) (26.5 g, 60.1mmol). The resulting solution was stirred at rt for 1 h. It was used forthe next reaction step directly without purification. LCMS (ES, m/z):440.2 [M+H]⁺.

Step 2:2-amino-9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert-butyldimethylsilyl)oxy)tetrahydro-4H-thieno[3,2-d][1,3,2]dioxasilin-6-yl)-1,9-dihydro-6H-purin-6-one

To the reaction mixture from the previous step at 0° C. was added1H-imidazole (17.05 g, 251 mmol) in one portion. The mixture was stirredrt for 0.5 h. tert-butylchlorodimethylsilane (15.10 g, 100 mmol) wasadded to the mixture, and it was stirred at 60° C. for 16 h. Then, thevolatile components were removed under reduced pressure. The solid wassuspended in cold methanol (75 mL), filtered, and washed with coldmethanol (2×30 mL). The solid was kept under reduced pressure to givethe product. LCMS (ES, m/z): 554.4 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆): δ10.80 (s, 1H), 7.98 (s, 1H), 6.49 (s, 2H), 5.53 (s, 1H), 4.46 (d, J=3.2Hz, 1H), 4.42 (d, J=9.9 Hz, 1H), 4.34 (dd, J=9.9, 4.7 Hz, 1H), 4.21 (t,J=10.5 Hz, 1H), 3.70-3.64 (m, 1H), 1.04 (s, 9H), 1.00 (s, 9H), 0.92 (s,9H), 0.19 (s, 3H), 0.11 (s, 3H).

Step 3:N-(9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert-butyldimethylsilyl)oxy)tetrahydro-4H-thieno[3,2-d][1,3,2]dioxasilin-6-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

2-amino-9-((4aR,6R,7R,7aS)-2,2-di-tert-butyl-7-((tert-butyldimethylsilyl)oxy)tetrahydro-4H-thieno[3,2-d][1,3,2]dioxasilin-6-yl)-1H-purin-6(9H)-one(29.1 g, 52.5 mmol) was co-evaporated with dry pyridine (3×50 mL) andre-dissolved in pyridine (70 mL) and dichloromethane (210 mL). Themixture was charged with Ar and cooled to 0° C. To the mixture was addedisobutyryl chloride (11.20 g, 105 mmol). It was stirred at rt for 4 h.It was concentrated under reduced pressure. The solid was suspended incold methanol (100 mL), filtered, and washed with cold methanol (3×50mL). The solid was kept under reduced pressure to give the product. LCMS(ES, m/z): 624.1 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆): δ 12.13 (s, 1H),11.39 (s, 1H), 8.32 (s, 1H), 5.61 (s, 1H), 4.66 (d, J=3.4 Hz, 1H), 4.48(d, J=9.9 Hz, 1H), 4.38-4.33 (m, 1H), 4.21 (t, J=9.9 Hz, 1H), 3.76-3.70(m, 1H), 2.84-2.80 (m, 1H), 1.13 (d, J=6.7 Hz, 6H), 1.06 (s, 9H), 1.01(s, 9H), 0.91 (s, 9H), 0.16 (s, 3H), 0.11 (s, 3H).

Step 4.N-(9-((2R,3R,4S,5R)-3-((tert-butyldimethylsilyl)oxy)-4-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

HF-Pyridine (26.6 g, 188 mmol) at 0° C. was diluted with pyridine (29mL). The resulting solution was added slowly to a stirred suspension ofN-(9-((4aR,6R,7R,7aS)-2,2-di-tert-butyl-7-((tert-butyldimethylsilyl)oxy)tetrahydro-4H-thieno[3,2-d][1,3,2]dioxasilin-6-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide(29.3 g, 47.0 mmol) in CH₂Cl₂ (290 mL) at 0° C. It was stirred at 0° C.for 2 h. The reaction mixture was diluted with CH₂Cl₂ (500 mL). It waswashed with water (500 mL) and sat aq NaHCO₃ (500 mL). The organic layerwas dried (Na₂SO₄) and concentrated to give the product. LCMS (ES, m/z):484.4 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆): δ 12.05 (s, 1H), 11.73 (s, 1H),8.43 (s, 1H), 5.89 (d, J=7.9 Hz, 1H), 5.34 (d, J=3.9 Hz, 1H), 5.27 (t,J=5.6 Hz, 1H), 4.60 (dd, J=8.1, 3.2 Hz, 1H), 4.19-4.17 (m, 1H),3.80-3.74 (m, 1H), 3.66-3.60 (m, 1H), 3.30 (t, J=8.0 Hz, 1H), 2.80-2.73(m, 1H), 1.12 (d, J=6.7 Hz, 6H), 0.68 (s, 9H), −0.06 (s, 3H), −0.29 (s,3H).

Step 5.N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)-4-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

N-(9-((2R,3R,4S,5R)-3-((tert-butyldimethylsilyl)oxy)-4-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide(21 g, 43.4 mmol) was co-evaporated with pyridine (3×50 mL) anddissolved in pyridine (210 mL).4,4′-(chloro(phenyl)methylene)bis(methoxybenzene) (16.2 g, 47.8 mmol)was added, and it was stirred at rt for 3 h and then concentrated underreduced pressure and co-evaporated with toluene (3×50 mL). The crude waspurified by silica gel chromatography eluting with 0-40% EtOAc in CH₂Cl₂(containing 0.1% Et₃N) to give the product. LCMS (ES, m/z): 786.4[M+H]⁺. ¹H-NMR (300 MHz, DMSO-d₆): δ 12.06 (s, 1H), 11.71 (s, 1H), 8.19(s, 1H), 7.44-7.42 (m, 2H), 7.37-7.22 (m, 7H), 6.92 (d, J=8.5 Hz, 4H),5.87 (d, J=7.2 Hz, 1H), 5.44 (d, J=4.4 Hz, 1H), 4.40 (dd, J=7.3, 3.3 Hz,1H), 4.19 (d, J=5.9 Hz, 1H), 3.75 (s, 6H), 3.54-3.35 (m, 2H), 3.34-3.28(m, 1H), 2.83-2.71 (m, 1H), 1.11 (d, J=6.7 Hz, 6H), 0.70 (s, 9H), −0.08(s, 3H), −0.29 (s, 3H).

Step 6:N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)-4-fluorotetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

To a solution ofN-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)-methyl)-3-((tert-butyldimethylsilyl)oxy)-4-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide(22 g, 28.0 mmol) in CH₂Cl₂ (220 mL) at 0° C. was added pyridine (18.11mL, 224 mmol) and DAST (14.79 mL, 112 mmol) dropwise. The reaction wasallowed to warm to rt and stirred for 7 h. It was then cooled to 0° C.and quenched by slow addition of sat aq NaHCO₃ (500 mL). More CH₂Cl₂(500 mL) was added, and the phases were separated. The organic phase waswashed with sat aq NaHCO₃ (3×200 mL) and brine (300 mL), dried (Na₂SO₄),concentrated, and purified by reverse phase (C18) chromatography elutingwith 45-95% of ACN in aq NH₄HCO₃ (5 mM) to give the product. LCMS (ES,m/z): 788.2 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆): δ 12.06 (s, 1H), 11.56(s, 1H), 8.02 (s, 1H), 7.44-7.42 (m, 2H), 7.34-7.23 (m, 7H), 6.92-6.89(m, 4H), 5.71 (d, J=4.9 Hz, 1H), 5.14 (dt, J=51.0, 5.6 Hz, 1H),5.01-4.96 (m, 1H), 3.88-3.85 (m, 1H), 3.75 (s, 6H), 3.57 (t, J=8.8 Hz,1H), 3.50 (dd, J=10.0, 5.3 Hz, 1H), 2.81-2.74 (m, 1H), 1.12 (d, J=6.8Hz, 6H), 0.77 (s, 9H), 0.00 (s, 3H), −0.16 (s, 3H). F-NMR: (376 MHz,DMSO-d₆, ppm) 6-193.99 (s, 1F).

Step 7:N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

To a solution of N-(9-((2R,3R,4R,SR)-5-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)-4-fluorotetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide(7.2 g, 6.85 mmol) in THF (70 mL) at rt was added TBAF (1.0M in THF,8.22 mL, 8.22 mmol) dropwise. It was stirred at rt for 30 min, thesolution was concentrated, and CH₂Cl₂ (300 mL) was added. The mixturewas washed with NaHCO₃ (3×200 mL) and brine (200 mL), and the organicphase was separated, dried (Na₂SO₄), concentrated and purified byreverse phase (C18) chromatography eluting with 0-95% of ACN in aqNH₄HCO₃ (5 mM) to give the product. LCMS (ES, m/z): 674.3 [M+H]⁺. ¹H-NMR(400 MHz, DMSO-d₆): δ 12.02 (br, 1H), 7.85 (s, 1H), 7.44-7.41 (m, 2H),7.36-7.24 (m, 7H), 6.94-6.90 (m, 4H), 6.33 (bs, 1H), 5.78 (d, J=2.7 Hz,1H), 5.19 (dt, J=50.2, 4.0 Hz, 1H), 4.80-4.75 (m, 1H), 4.10-4.02 (m,1H), 3.76 (s, 6H), 3.55 (dd, J=9.3, 5.6 Hz, 1H), 3.44 (t, J=8.9 Hz, 1H),2.79-2.71 (m, 1H), 1.12 (d, J=6.8 Hz, 6H). F-NMR: (376 MHz, DMSO-d₆) δ−194.75 (s).

Preparation 18:N-(9-((2R,3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

Step 1: (3R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-3-yl2,4-dimethoxybenzoate

To a solution of(6aR,9R,9aS)-2,2,4,4-tetraisopropyltetrahydro-6H-thieno[3,2-f][1,3,5,2,4]trioxadisilocin-9-yl2,4-dimethoxybenzoate (60 g, 108 mmol) in THF (500 mL) were added AcOH(13.59 g, 226 mmol) and TBAF in THF (1 M, 226 mL, 226 mmol). After 1 h,it was concentrated under reduced pressure and purified by silica gelcolumn chromatography eluting with 0-20% EtOAc in CH₂Cl₂ to give theproduct. LCMS (ES, m/z): 315.1 [M+H]⁺. 1H-NMR (400 MHz, CDCl₃) δ 7.87(d, J=8.7 Hz, 1H), 6.58-6.46 (m, 2H), 5.51 (dt, J=5.0, 3.7 Hz, 1H), 4.31(td, J=6.9, 3.7 Hz, 1H), 3.88 (d, J=12.0 Hz, 8H), 3.58 (dt, J=7.1, 4.7Hz, 1H), 3.26 (dd, J=12.2, 5.0 Hz, 1H), 3.15-3.01 (m, 2H), 2.31 (s, 1H).

Step 2:(3R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-hydroxytetrahydrothiophen-3-yl2,4-dimethoxybenzoate

To a solution of(3R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-3-yl2,4-dimethoxybenzoate (33 g, 105 mmol) in pyridine (300 mL) was addedtert-butylchlorodiphenylsilane (43.3 g, 157 mmol). It was stirred at rtfor 4 h. Then, water (300 mL) was added. Layers were separated, and theaq layer was extracted with CH₂Cl₂ (3×300 mL). The combined organiclayer was washed with brine (300 mL), dried (Na₂SO₄), concentrated, andpurified by silica gel column chromatography eluting with 1 to 10% EtOAcin petroleum ether to give the product. LCMS (ES, m/z): 575.3 [M+Na]⁺.¹H-NMR (400 MHz, DMSO-d₆) δ 7.83 (d, J=8.7 Hz, 1H), 7.68 (t, J=5.5 Hz,5H), 7.54-7.38 (m, 6H), 6.69-6.60 (m, 2H), 5.40 (d, J=5.6 Hz, 1H),5.35-5.29 (m, 1H), 4.12 (s, 1H), 4.05 (d, J=7.3 Hz, 1H), 3.85 (d, J=7.2Hz, 6H), 3.78-3.66 (m, 1H), 3.55 (d, J=7.2 Hz, 1H), 3.14 (dd, J=11.0,5.0 Hz, 1H), 2.86-2.77 (m, 1H), 1.03 (s, 9H).

Step 3:(3R,4S,5R)-4-((1H-imidazole-1-carbonothioyl)oxy)-5-(((tert-butyldiphenylsilyl)oxy)methyl)tetrahydrothiophen-3-yl2,4-dimethoxybenzoate

To a solution of(3R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-hydroxytetrahydrothiophen-3-yl2,4-dimethoxybenzoate (48 g, 87 mmol) in dichloroethane (500 mL) wasadded di(1H-imidazol-1-yl)methanethione (20.12 g, 113 mmol). It washeated at 85° C. under Ar for 1 h. Then, it was concentrated and used inthe next step without purification. LCMS (ES, m/z): 663.2 [M+H]⁺.

Step 4:(3R,5S)-5-(((tert-butyldiphenylsilyl)oxy)methyl)tetrahydrothiophen-3-yl2,4-dimethoxybenzoate

To a solution of(3R,4S,5R)-4-((1H-imidazole-1-carbonothioyl)oxy)-5-(((tert-butyldiphenylsilyl)oxy)methyl)tetrahydrothiophen-3-yl2,4-dimethoxybenzoate (crude, 57.6 g, 87 mmol) in THF (40 mL) andtoluene (200 mL) was added tributylstannane (139 g, 478 mmol). It washeated at 95° C., and 2,2′-(diazene-1,2-diyl)bis(2-methylpropanenitrile)(1.427 g, 8.69 mmol) in toluene (200 mL) was added over 30 min. After 1h, the resulting mixture was concentrated and purified by silica gelcolumn chromatography eluting with 0 to 10% EtOAc in petroleum ether togive the product. LCMS (ES, m/z): 537.3 [M+H]⁺. ¹H-NMR (400 MHz, CDCl₃)δ 7.88 (d, J=8.6 Hz, 1H), 7.77-7.67 (m, 4H), 7.50-7.36 (m, 6H),6.58-6.48 (m, 2H), 5.70 (p, J=3.8 Hz, 1H), 3.95-3.74 (m, 10H), 3.28 (dd,J=12.0, 4.6 Hz, 1H), 3.10-3.02 (m, 1H), 2.49-2.39 (m, 1H), 1.92 (ddd,J=13.3, 8.6, 4.1 Hz, 1H), 1.09 (s, 9H).

Step 5: (JR,3R,5S)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-1-oxidotetrahydrothiophen-3-yl2,4-dimethoxybenzoate

To a solution of Ti(OiPr)₄ (23.29 mL, 78 mmol) in CH₂Cl₂ (130 mL) underAr was added diethyl (L)-tartrate (38.3 mL, 224 mmol) dropwise. After 10min, the mixture was cooled to −20° C., and then TBHP in decane (˜5.5M,27.1 mL, 149 mmol) was added dropwise. After 5 min, a solution of (3R,5S)-5-(((tert-butyldiphenylsilyl)oxy)methyl)tetrahydrothiophen-3-yl2,4-dimethoxybenzoate (40 g, 74.5 mmol) in CH₂Cl₂ (130 mL) was added tothe reaction at −20° C. The resulting mixture was stirred at −20° C. for16 h. It was quenched by the addition of ice water (300 mL) and allowedto warm up to rt. The precipitate was filtered off and washed with EtOAc(3×300 mL). The filtrate was washed with water (3×200 mL). The aq layerwas extracted with EtOAc (400 mL). The combined organic layer was dried(Na₂SO₄), concentrated and purified by flash chromatography eluting with0 to 70% EtOAc in petroleum ether to give the product (mixture of twoisomers). LCMS (ES, m/z): 553.2 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆) δ 7.81(d, J=8.7 Hz, 1H), 7.75-7.61 (m, 4H), 7.54-7.39 (m, 6H), 6.67-6.56 (m,2H), 5.66 (q, J=3.8 Hz, 1H), 4.12 (dt, J=10.5, 4.4 Hz, 1H), 3.92-3.79(m, 8H), 3.58-3.42 (m, 1H), 3.20-3.09 (m, 1H), 2.97 (d, J=15.0 Hz, 1H),2.05 (ddd, J=14.5, 10.5, 4.3 Hz, 1H), 1.02 (s, 9H).

Step 6:(3R,5S)-2-acetoxy-5-(((tert-butyldiphenylsilyl)oxy)methyl)tetrahydrothiophen-3-yl2,4-dimethoxybenzoate

A solution of(3R,5S)-2-acetoxy-5-(((tert-butyldiphenylsilyl)oxy)methyl)-tetrahydrothiophen-3-yl2,4-dimethoxybenzoate (21 g, 34.2 mmol) in acetic anhydride (210 mL) washeated at 110° C. After stirring of 3.5 h, the reaction mixture wascooled to rt and concentrated. The residue was purified by silica gelcolumn chromatography eluting with 0% to 20% EtOAc in petroleum ether togive the product. LCMS (ES, m/z): 535.3 [M-OAc]⁺. ¹H-NMR (400 MHz,DMSO-d₆) δ 7.76-7.61 (m, 5H), 7.46 (dq, J=7.6, 4.5, 3.7 Hz, 6H),6.68-6.58 (m, 2H), 6.22 (d, J=4.3 Hz, 0.65H), 5.94 (s, 0.28H), 5.51-5.46(m, 0.33H), 5.38 (ddd, J=11.1, 7.2, 4.3 Hz, 0.65H), 3.96-3.61 (m, 9H),2.40-2.21 (m, 2H), 2.03 (d, J=1.6 Hz, 3H), 1.01 (s, 9H).

Step 7:(3R,5S)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-2-(6-chloro-2-isobutyramido-9H-purin-9-yl)tetrahydrothiophen-3-yl2,4-dimethoxybenzoate

To a solution of N-(6-chloro-9H-purin-2-yl)isobutyramide (10.27 g, 42.9mmol) in toluene (600 mL) at 0° C. was added trimethylsilylN-(trimethylsilyl)acetimidate (23.26 g, 114 mmol). It was heated at 80°C. for 1 h and was cooled to 0° C. again. To the reaction was then addeda solution of(3R,5S)-2-acetoxy-5-(((tert-butyldiphenylsilyl)oxy)methyl)-tetrahydrothiophen-3-yl2,4-dimethoxybenzoate (17 g, 28.6 mmol) in toluene (600 mL) andtrimethylsilyl trifluoromethanesulfonate (19.06 g, 86 mmol). It washeated to 80° C. and stirred under Ar for 12 h. At that time, thereaction was cooled to rt, and sat aq NaHCO₃ (400 mL) was added. Layerswere separated, and the aq layer was extracted with EtOAc (4×1000 mL).The combined organic phase was dried (Na₂SO₄), concentrated and purifiedby silica gel column chromatography eluting with 10% to 40% EtOAc inpetroleum ether to give the product (mixture of a and 3 isomers). LCMS(ES, m/z): 774.3 [M+H]⁺. ¹H-NMR (400 MHz, CDCl₃) δ 8.48 (s, 0.32H), 8.35(s, 0.69H), 7.97-7.83 (m, 1.67H), 7.70 (dq, J=8.4, 1.5 Hz, 4H), 7.54 (d,J=8.7 Hz, 0.33H), 7.49-7.36 (m, 6H), 6.57-6.36 (m, 2.5H), 6.18 (d, J=2.4Hz, 0.7H), 5.87-5.80 (m, 0.35H), 5.73 (q, J=3.3 Hz, 0.75H), 4.23-4.01(m, 1.2H), 3.98-3.74 (m, 7.8H), 3.11 (s, 0.73H), 2.95 (s, 0.37H),2.57-2.36 (m, 1.75H), 2.30-2.20 (m, 0.34H), 1.23 (d, J=6.8 Hz, 2.19H),1.19 (dd, J=6.8, 3.5 Hz, 4.38H), 1.09 (d, J=1.7 Hz, 9H).

Step 8.(2R,3R,5S)-2-(6-chloro-2-isobutyramido-9H-purin-9-yl)-5-(hydroxymethyl)-tetrahydrothiophen-3-yl2,4-dimethoxybenzoate

To a solution of (3R,5S)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-2-(6-chloro-2-isobutyramido-9H-purin-9-yl)tetrahydrothiophen-3-yl2,4-dimethoxybenzoate (20 g, 25.8 mmol) in THF (135 mL) was added TBAFin THF (1 M, 31 mL, 31 mmol) dropwise. After 1 h, the reaction mixturewas concentrated and purified by column chromatography using 1% to 10%MeOH in CH₂Cl₂ as the eluent to give a mixture of two isomers. It wasre-purified by reverse phase (C18) chromatography eluting with 10 to 45%ACN in aq NH₄CO₃ (5 mM) to give the product. LCMS (ES, m/z): 536.2[M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆) δ 10.80 (s, 1H), 8.83 (s, 1H), 7.72(d, J=8.7 Hz, 1H), 6.65-6.55 (m, 2H), 6.18 (d, J=2.5 Hz, 1H), 5.80 (q,J=3.5 Hz, 1H), 5.22 (t, J=5.1 Hz, 1H), 3.93-3.67 (m, 9H), 2.85 (p, J=6.9Hz, 1H), 2.70 (ddd, J=13.4, 8.5, 4.5 Hz, 1H), 2.36 (dt, J=14.1, 5.0 Hz,1H), 1.06 (dd, J=6.8, 3.3 Hz, 6H).

Step 9:2-amino-9-((2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-1,9-dihydro-6H-purin-6-one

To a solution of (2R,3R,5S)-2-(6-chloro-2-isobutyramido-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrothiophen-3-yl2,4-dimethoxybenzoate (6.0 g, 11.19 mmol) in MeOH (300 mL) were added2-mercaptoethanol (3.50 g, 44.8 mmol) and NaOMe (10.08 g, 56 mmol, 30%in MeOH). It was heated at 60° C. for 16 h, cooled to rt, and conc. HCl(4 mL) was added. The resulting mixture was concentrated, and water (100mL) and EtOAc (100 mL) were added. Layers were separated, and the aqlayer was extracted with EtOAc (3×100 mL). The aq layer was basifiedwith NaHCO₃ (solid) to ˜pH 8 and stirred at rt for 1 h. The precipitatewas filtered and kept under reduced pressure to give the product. LCMS(ES, m/z): 284.1 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆) δ 10.57 (s, 1H), 7.98(s, 1H), 6.46 (s, 2H), 5.57 (dd, J=10.9, 3.9 Hz, 2H), 5.12 (t, J=5.4 Hz,1H), 4.48 (p, J=4.1 Hz, 1H), 3.78-3.53 (m, 3H), 2.11-1.99 (m, 2H).

Step 10:N-(9-((2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

2-amino-9-((2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-1,9-dihydro-6H-purin-6-one(580 mg, 2.047 mmol) was co-evaporated with pyridine (3×20 mL) and thenre-dissolved in pyridine (20 mL). The mixture was cooled to 0° C. andthen treated with chlorotrimethylsilane (1557 mg, 14.33 mmol). It waswarmed to rt and stirred for 2 h. Then, the reaction was cooled to 0° C.again, and isobutyric anhydride (486 mg, 3.07 mmol) was added dropwise.It was warmed to rt and stirred for 2 h. The reaction was quenched bythe addition of methanol (5 mL). After 5 min, NH₄OH (ca 29%, 10 mL) wasadded. The mixture was stirred at rt for 30 min. Then, it wasconcentrated and purified by column chromatography on silica gel elutingwith 10% to 20% MeOH in CH₂Cl₂ to give the product. LCMS (ES, m/z):354.1 [M+H]⁺. ¹H-NMR (300 MHz, CD₃OD) δ: 8.40 (s, 1H), 5.83-5.82 (m,1H), 4.62-4.59 (m, 1H), 3.89-3.80 (m, 2H), 3.79-3.74 (m, 1H), 2.73-2.64(m, 1H), 2.19-2.11 (m, 2H), 1.20 (d, J=6.8 Hz, 6H).

Step 11:N-(9-((2R,3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

N-(9-((2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide(510 mg, 1.443 mmol) was co-evaporated with pyridine (3×5 mL) and thenre-suspended in pyridine (7 mL). To the suspension was added4,4′-(chloro(phenyl)methylene)bis(methoxybenzene) (538 mg, 1.587 mmol),and the mixture was stirred at rt for 2 h. At that time the mixture wasconcentrated under reduced pressure, and the residue was purified bycolumn chromatography on silica gel eluting with 1% to 4% MeOH in CH₂Cl₂(containing 1% Et₃N) to give the product. LCMS (ES, m/z): 656.0 [M+H]⁺.¹H-NMR (400 MHz, CD₃OD) δ: 8.02 (s, 1H), 7.51-7.43 (m, 2H), 7.40-7.17(m, 7H), 6.91-6.82 (m, 4H), 5.85-5.84 (d, J=2.3 Hz, 1H), 4.59-4.57 (m,1H), 4.02-3.95 (m, 1H), 3.78 (s, 6H), 3.52-3.34 (m, 3H), 2.72-2.68 (m,1H), 1.95-191 (m, 1H), 1.38 (d, J=6.8 Hz, 6H).

Preparation 19:N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-fluoro-3-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

Step 1:((2R,3R,4S)-4-(benzoyloxy)-3-fluoro-5-hydroxytetrahydrofuran-2-yl)methylbenzoate

To a stirred solution of((2R,3R,4S)-5-acetoxy-4-(benzoyloxy)-3-fluorotetrahydrofuran-2-yl)methylbenzoate (20.0 g, 49.7 mmol) in dry 1,2-dichloroethane (200 mL) wasadded tri-N-butyltin methoxide (28.8 mL, 99 mmol). The resulting mixturewas stirred at 80° C. for 3 h and then concentrated in vacuo. Theresidue was diluted in 500 mL of ethyl acetate and washed with sat aq.NH₄Cl (500 mL) and brine (500 mL). The separated organic layer was driedover anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residuewas purified by a silica gel column, eluting with 0-40% EtOAc/Hexane.LCMS (ES, m/z): 343.2 [M+H-H₂O]⁺. ¹H NMR (500 MHz, Chloroform-d) δ8.18-8.00 (m, 7H), 7.68-7.57 (m, 3H), 7.61-7.35 (m, 7H), 5.73 (dd,J=8.3, 4.6 Hz, 1H), 5.65 (dt, J=3.9, 2.0 Hz, 1H), 5.54 (t, J=4.7 Hz,0H), 5.50-5.40 (m, 2H), 5.36-5.24 (m, 1H), 4.87 (dtd, J=25.5, 4.0, 1.5Hz, 1H), 4.74-4.46 (m, 4H), 4.15 (q, J=7.2 Hz, 1H), 3.39 (d, J=4.1 Hz,1H), 3.30 (dd, J=8.6, 3.4 Hz, 1H), 2.07 (s, 2H), 1.41-1.23 (m, 3H).

Step 2. (2R,3R,4S)-3-fluoro-2-hydroxy-5-(methoxyimino)pentane-1,4-diyldibenzoate

To a stirred solution of((2R,3R,4S)-4-(benzoyloxy)-3-fluoro-5-hydroxytetrahydrofuran-2-yl)methylbenzoate (17.9 g, 49.7 mmol) in dry MeOH (100 mL) was addedO-methylhydroxylamine hydrochloride (6.23 g, 74.6 mmol), followed bytriethylamine (10.39 mL, 74.6 mmol). The reaction mixture was stirred atrt for 2 h and then concentrated in vacuo. The residue was diluted in500 mL of ethyl acetate and washed with sat aq. NH₄Cl (500 mL) and brine(500 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered,and concentrated in vacuo. The residue was used for the next stepdirectly without further purification. LCMS (ES, m/z): 390.2 [M+H]⁺.

Step 3:(2R,3S,4S)-3-fluoro-5-(methoxyimino)-2-(((4-nitrophenyl)sulfonyl)oxy)pentane-1,4-diyldibenzoate

To a stirred solution of(2R,3R,4S)-3-fluoro-2-hydroxy-5-(methoxyimino)pentane-1,4-diyldibenzoate (19.4 g, 49.7 mmol) in dry EtOAc (100 mL) was added4-nitrobenzenesulfonyl chloride (16.5 g, 74.6 mmol), followed bytriethylamine (10.4 mL, 74.6 mmol). The reaction mixture was stirred atrt for 18 h and was then diluted with 200 mL of ethyl acetate, washedwith water (300 mL) and brine (300 mL). The separated organic layer wasdried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. Theresidue was purified by a silica gel column, eluting with 0-30%EtOAc/Hexane. LCMS (ES, m/z): 575.3 [M+H]⁺. ¹H NMR (500 MHz,Chloroform-d) δ 8.25-7.99 (m, 7H), 7.94-7.80 (m, 2H), 7.68-7.55 (m, 2H),7.57-7.46 (m, 3H), 7.50-7.38 (m, 3H), 6.55 (ddd, J=17.2, 5.3, 2.8 Hz,0H), 5.89 (ddd, J=17.7, 6.2, 4.1 Hz, 1H), 5.46-5.37 (m, 1H), 5.40-5.28(m, 1H), 5.21 (t, J=4.3 Hz, 0H), 4.84 (tdd, J=12.8, 2.6, 1.5 Hz, 1H),4.53 (dddd, J=23.1, 13.0, 7.0, 1.8 Hz, 1H), 4.14 (q, J=7.2 Hz, 1H), 3.99(d, J=36.3 Hz, 4H), 2.06 (s, 2H), 1.44-1.23 (m, 2H).

Step 4. (2S,3S,4S)-2-bromo-3-fluoro-5-(methoxyimino)pentane-1,4-diyldibenzoate

To a stirred solution of (2R,3S,4S)-3-fluoro-5-(methoxyimino)-2-(((4-nitrophenyl)sulfonyl)oxy)pentane-1,4-diyldibenzoate (21.3 g, 37.1 mmol) in dry DMF (100 mL) was added freshlyopened lithium bromide powder (16.1 g, 185 mmol). The resulting mixturewas stirred at 60° C. for 18 h. The reaction mixture was diluted in 300mL of ethyl acetate and washed with water (500 mL) and brine (500 mL).The separated organic layer was dried over anhydrous Na₂SO₄, filtered,and concentrated in vacuo. The residue was purified by a silica gelcolumn, eluting with 0-25% EtOAc/Hexane to give product. LCMS (ES, m/z):452.1 [M+H]⁺. ¹H NMR (500 MHz, Chloroform-d) δ 8.15-8.03 (m, 11H),7.69-7.56 (m, 8H), 7.56-7.41 (m, 12H), 6.93-6.86 (m, 1H), 6.45 (ddd,J=14.7, 5.9, 4.7 Hz, 0H), 5.93 (ddd, J=11.8, 6.7, 5.9 Hz, 2H), 5.32 (s,1H), 5.29-5.10 (m, 3H), 4.89-4.77 (m, 3H), 4.80-4.67 (m, 3H), 4.60-4.40(m, 3H), 3.95 (d, J=16.9 Hz, 2H), 3.89 (s, 6H), 1.32-1.22 (m, 1H).

Step 5. (2S,3S,4S)-2-bromo-3-fluoro-5-oxopentane-1,4-diyl dibenzoate

To a stirred solution of(2S,3S,4S)-2-bromo-3-fluoro-5-(methoxyimino)pentane-1,4-diyl dibenzoate(20.0 g, 44.2 mmol) in THF (200 mL) was added 37% aqueous solution offormaldehyde (32.9 mL, 442 mmol) and 1N HCl (44.2 mL, 44.2 mmol). Theresulting mixture was stirred at 55° C. for 5 h. The reaction mixturewas concentrated in vacuo to remove most of the THF. The residue wasdiluted in 300 mL of ethyl acetate and washed with water (300 mL) andbrine (300 mL). The separated organic layer was dried over anhydrousNa₂SO₄, filtered, and concentrated in vacuo. The residue was used forthe next step without further purification. LCMS (ES, m/z): 423.2[M+H]⁺.

Step 6:((2R,3S,4R)-4-(benzoyloxy)-3-fluoro-5-hydroxytetrahydrothiophen-2-yl)methylbenzoate

To a stirred solution of(2S,3S,4S)-2-bromo-3-fluoro-5-oxopentane-1,4-diyl dibenzoate (18.7 g,44.2 mmol) in NMP (150 mL) at 0° C. was added sodium hydrosulfide (5.0g, 89.0 mmol). The resulting mixture was stirred at 0° C. for 30 min andthen at rt for 30 min. The reaction mixture was diluted in 300 mL ofethyl acetate and washed with water (300 mL) and brine (300 mL). Theorganic layer was dried over anhydrous Na₂SO₄, filtered, andconcentrated in vacuo. The residue was used for the next step withoutfurther purification. LCMS (ES, m/z): 359.2 [M+H—H₂O]⁺.

Step 6:((2R,3S,4R)-5-acetoxy-4-(benzoyloxy)-3-fluorotetrahydrothiophen-2-yl)methylbenzoate

To a stirred solution of ((2R,3S,4R)-4-(benzoyloxy)-3-fluoro-5-hydroxytetrahydrothiophen-2-yl)methylbenzoate (16.6 g, 44.2 mmol) in dry THF (150 mL) at 0° C. was addedacetic anhydride (8.3 mL, 133 mmol) and trimethylamine (18.5 mL, 133mmol). The resulting mixture was stirred at 0° C. for 30 min and then atrt for 2 h. The reaction was quenched by addition of MeOH, and thereaction mixture was concentrated in vacuo to remove most of the THF.The residue was diluted in 300 mL of ethyl acetate and washed with water(300 mL) and brine (300 mL). The separated organic layer was dried overanhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue waspurified by a silica gel column, eluting with 0-25% EtOAc/Hexane to giveproduct. LCMS (ES, m/z): 441.2 [M+Na]⁺. ¹H NMR (500 MHz, Chloroform-d) δ8.15-8.02 (m, 4H), 7.68-7.57 (m, 2H), 7.55-7.42 (m, 4H), 6.02 (t, J=2.4Hz, 1H), 5.88 (ddd, J=6.1, 3.6, 2.4 Hz, 1H), 5.50-5.42 (m, 0H), 5.35(dd, J=7.3, 3.6 Hz, 0H), 5.32 (s, 0H), 4.80-4.69 (m, 1H), 4.56 (dd,J=11.6, 5.9 Hz, 1H), 4.26-4.10 (m, 2H), 2.25-2.11 (m, 1H), 2.08 (d,J=10.0 Hz, 3H), 1.29 (t, J=7.1 Hz, 1H).

Step 7:((2R,3S,4R,5R)-4-(benzoyloxy)-3-fluoro-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-2-yl)methylbenzoate

To a suspension of N-(6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide(15.86 g, 71.7 mmol) in ClCH₂CH₂Cl (300 mL) was added (Z)-trimethylsilylN-(trimethylsilyl)acetimidate (35.1 mL, 143 mmol). The suspension wasstirred at 70° C. overnight and was then cooled to −15° C. To thismixture was added((2R,3S,4R)-5-acetoxy-4-(benzoyloxy)-3-fluorotetrahydrothiophen-2-yl)methylbenzoate (10 g, 23.90 mmol), followed by TMS-OTf (8.64 mL, 47.8 mmol).The reaction mixture was stirred at −15° C. for 2 h, then at rt for 5 hand finally at 70° C. for 5d. The reaction mixture was allowed to coolto RT and was then filtered. The filtrate was washed with sat. aq.NaHCO₃, brine and then dried over Na₂SO₄ and concentrated in vacuo. Theresidue was purified by silica gel column, eluting with 0-60%EtOAc/Hexane. Subsequent recrystallization from EtOAc gave product. LCMS(ES, m/z): 580.5 [M+H]⁺. ¹H NMR (500 MHz, Chloroform-d) δ 12.19 (s, 0H),9.56 (s, 1H), 8.12-8.06 (m, 1H), 8.02-7.96 (m, 1H), 7.66 (s, 0H),7.71-7.58 (m, 1H), 7.57-7.50 (m, 1H), 7.54-7.42 (m, 1H), 6.59 (ddd,J=25.3, 7.4, 3.1 Hz, 1H), 6.20 (d, J=7.4 Hz, 0H), 5.68-5.59 (m, 1H),4.87 (ddd, J=11.7, 7.7, 1.5 Hz, 1H), 4.26-4.11 (m, 1H), 2.96 (hept,J=6.8 Hz, 1H), 1.38 (dd, J=19.0, 6.9 Hz, 3H).

Step 8:N-(9-((2R,3R,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

To a solution of ((2R,3S,4R,5R)-4-(benzoyloxy)-3-fluoro-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-2-yl)methylbenzoate (0.96 g, 1.66 mmol) in THF (5 mL)/MeOH (4 mL)/H₂O (1 mL) at 0°C. was added 2N sodium hydroxide (1.8 mL, 3.6 mmol). The reactionmixture was stirred at 0° C. for 30 min and then neutralized with aceticacid (0.38 mL, 6.6 mmol). The product was collected by filtration andcarried on to the next step without further purification. LCMS (ES,m/z): 372.3 [M+H]⁺.

Step 9:N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

To a solution ofN-(9-((2R,3R,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)-tetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide(0.62 g, 1.66 mmol) in pyridine (25 mL) at 0° C. was added4,4′-dimethoxytrityl chloride (0.84 g, 2.48 mmol). The reaction mixturewas stirred at 0° C. for 3 h. The reaction was quenched with H₂O (1 mL),and the mixture was concentrated. The residue was diluted in 100 mL ofethyl acetate and washed with saturated aq. NaHCO₃ (100 mL) and brine(100 mL). The separated organic layer was dried over anhydrous Na₂SO₄,filtered, and concentrated in vacuo. The residue was purified by asilica gel column, eluting with 0-100% EtOAc/Hexane containing 0.1% Et₃Nto give product. LCMS (ES, m/z): 674.6 [M+H]⁺. ¹H NMR (500 MHz,Chloroform-d) δ 11.96 (s, 0H), 8.35 (s, 0H), 7.70 (s, 0H), 7.63-7.56 (m,1H), 7.50-7.43 (m, 2H), 7.38-7.24 (m, 1H), 6.93-6.85 (m, 2H), 5.92 (d,J=8.3 Hz, 0H), 5.35-5.23 (m, 1H), 4.15 (q, J=7.1 Hz, 1H), 3.85-3.69 (m,3H), 3.46 (dd, J=10.2, 5.4 Hz, 0H), 3.35 (dd, J=10.2, 5.2 Hz, 0H), 2.09(d, J=19.4 Hz, 2H), 1.34-1.22 (m, 2H), 1.01 (d, J=6.8 Hz, 1H), 0.91 (d,J=6.9 Hz, 1H).

Preparation 20:N-(3-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-fluoro-3-hydroxytetrahydrothiophen-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

Step 1:N-(7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

To a suspension of5-amino-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (5.0 g,32.9 mmol) in anhydrous DMF (60 mL) was added isobutyric anhydride (12.5mL, 76.0 mmol) dropwise. The reaction mixture was refluxed at 150° C.for 1 h. The reaction was quenched with MeOH (6.6 mL, 164 mmol) andconcentrated in vacuo. The residue was taken up in DCM (50 mL)/Hexane(100 mL) and was stirred at vigorously at rt for 15 min. The product wascollected by filtration. LCMS (ES, m/z): 223.2 [M+H]⁺. ¹H NMR (500 MHz,DMSO-d₆) δ 16.04 (s, 1H), 12.19 (s, 1H), 11.78 (s, 1H), 2.78 (hept,J=6.7 Hz, 1H), 1.13 (d, J=6.8 Hz, 6H).

Step 2:((2R,3S,4R,5R)-4-(benzoyloxy)-3-fluoro-5-(5-isobutyramido-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)tetrahydrothiophen-2-yl)methylbenzoate

To a mixture of ((2R,3S,4R)-5-acetoxy-4-(benzoyloxy)-3-fluorotetrahydrothiophen-2-yl)methylbenzoate (1.5 g, 3.6 mmol) andN-(7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide(0.96 g, 4.3 mmol) in nitromethane (20 mL) was added BF₃*Et₂O (0.54 mL,4.3 mmol) and the resulting mixture was heated at 100° C. undermicrowave irradiation for 1 h. The reaction mixture was cooled, dilutedin 100 mL of ethyl acetate and washed with saturated aq. NaHCO₃ (100 mL)and brine (100 mL). The organic portion was dried over Na₂SO₄ andconcentrated in vacuo. The residue was purified by a silica gel column,eluting with 0-35% EtOAc/Hexane. LCMS (ES, m/z): 581.4 [M+H]⁺. ¹H NMR(500 MHz, Chloroform-d) δ 12.27 (s, 2H), 9.73 (s, 2H), 8.19-7.93 (m,8H), 7.86-7.80 (m, 0H), 7.71-7.55 (m, 4H), 7.57-7.39 (m, 8H), 7.40-7.33(m, 0H), 6.76 (d, J=6.7 Hz, 2H), 6.62-6.48 (m, 2H), 5.79 (t, J=2.9 Hz,1H), 5.69 (t, J=2.9 Hz, 1H), 5.62-5.54 (m, 2H), 4.89 (ddd, J=11.6, 7.8,1.3 Hz, 2H), 4.78-4.65 (m, 1H), 4.31-4.18 (m, 2H), 4.15 (q, J=7.1 Hz,3H), 2.95 (hept, J=6.9 Hz, 2H), 2.81-2.68 (m, 1H), 2.07 (s, 4H), 1.39(dd, J=17.5, 6.9 Hz, 10H), 1.34-1.24 (m, 8H).

Step 3:N-(3-((2R,3R,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

To a solution of ((2R,3S,4R,5R)-4-(benzoyloxy)-3-fluoro-5-(5-isobutyramido-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)tetrahydrothiophen-2-yl)methylbenzoate (4.5 g, 7.8 mmol) in THF (35 mL)/MeOH (28 mL)/H (7 mL) at 0° C.was added 2N sodium hydroxide (8.6 mL, 17.2 mmol). The reaction mixturewas stirred at 0° C. for 1 h and then neutralized with acetic acid (2.3mL, 39.0 mmol). Product was collected by filtration and carried on tothe next step without further purification. LCMS (ES, m/z): 373.3[M+H]⁺.

Step 4:N-(3-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrothiophen-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

To a solution ofN-(3-((2R,3R,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)-tetrahydrothiophen-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide(1.8 g, 4.8 mmol) in pyridine (30 mL) at 0° C. was added4,4′-dimethoxytrityl chloride (1.8 g, 5.3 mmol). The reaction mixturewas stirred at 0° C. for 1 h. The reaction was quenched with H₂O (1 mL),and the mixture was concentrated. The residue was diluted in 100 mL ofethyl acetate and washed with saturated aq. NaHCO₃ (100 mL) and brine(100 mL). The separated organic layer was dried over anhydrous Na₂SO₄,filtered, and concentrated in vacuo. The residue was purified by asilica gel column, eluting with 0-100% EtOAc/Hexane containing 0.1% Et₃Nto give product. LCMS (ES, m/z): 675.5 [M+H]⁺. ¹H NMR (500 MHz,Chloroform-d) δ 12.16 (s, 1H), 8.48 (s, 0H), 7.57-7.50 (m, 1H),7.49-7.37 (m, 2H), 7.39-7.29 (m, 0H), 7.31 (s, 1H), 7.32-7.14 (m, 1H),6.91-6.81 (m, 2H), 6.22-6.16 (m, 0H), 5.42-5.31 (m, 1H), 3.77 (d, J=36.0Hz, 7H), 3.46 (dd, J=10.2, 5.6 Hz, 0H), 3.36 (dd, J=10.2, 5.4 Hz, 0H),2.17 (p, J=6.9 Hz, 0H), 1.06 (dd, J=26.1, 6.9 Hz, 3H).

Preparation 21:N-(3-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-3,4-dihydroxytetrahydrothiophen-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

Step 1:(2R,3S,4R,5R)-2-((benzoyloxy)methyl)-5-(5-isobutyramido-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)tetrahydrothiophene-3,4-diyldibenzoate

BF₃.OEt₂ (3.65 mL, 28.8 mmol) was added dropwise to a mixture of(2R,3R,4S,5R)-2-acetoxy-5-((benzoyloxy)methyl)tetrahydrothiophene-3,4-diyldibenzoate (10.0 g, 19.2 mmol) andN-(7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5yl)isobutyramide (5.98 g, 26.9 mmol) in MeNO₂ (180 mL) at ambienttemperature. Upon completion of addition, the mixture was heated at 120°C. in a microwave reactor for 45 min. The sample was cooled to rt andconcentrated in vacuo. The residue was purified by flash columnchromatography eluting with EtOAc/isohexane (10-90%) to give desiredproduct. LCMS (ES, m/z): 683.5 [M+H]⁺. ¹H NMR (500 MHz, Chloroform-d) δ12.24 (s, 1H), 9.77 (s, 1H), 8.08-7.95 (m, 4H), 7.94-7.87 (m, 2H),7.70-7.35 (m, 9H), 6.84 (d, J=5.9 Hz, 1H), 6.67 (dd, J=5.9, 3.9 Hz, 1H),6.49 (t, J=3.7 Hz, 1H), 5.52 (dd, J=11.4, 7.8 Hz, 1H), 5.04 (dd, J=11.4,7.8 Hz, 1H), 4.28 (m, 1H), 2.97 (hept, J=6.9 Hz, 1H), 1.42 (dd, 6.9 Hz,6H).

Step 2:N-(3-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

To a stirred solution of the product from Step 1 (5.3 g, 7.8 mmol)dissolved in Pyridine (8 mL) and MeOH (32 mL) at 25° C. was added sodiumhydroxide (1.24 g, 31.1 mmol) in one portion. The mixture was stirred at25° C. for 15 min before the addition of acetic acid (1.8 mL, 31.1mmol). The mixture was concentrated in vacuo, and the residue waspurified by flash column chromatography eluting with EtOAc/isohexane(70-100%) to give the desired product. LCMS (ES, m/z): 371.3 [M+H]⁺. ¹HNMR (500 MHz, DMSO-d₆) δ 12.24 (s, 1H), 11.96 (s, 1H), 5.91 (d, J=5.6Hz, 1H), 5.73 (d, J=5.5 Hz, 1H), 5.43 (d, J=5.0 Hz, 1H), 5.12 (m, 1H),4.81 (m, br, 1H), 4.40 (m, 1H), 3.89-3.77 (m, 1H), 3.53 (m, 1H),3.46-3.36 (m, 1H), 2.79 (m, 1H), 1.14 (dd, J=6.7, 1.2 Hz, 6H).

Step 3:N-(3-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dihydroxytetrahydrothiophen-2-yl)-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)isobutyramide

To the product from step 2 (2.3 g, 6.2 mmol) was added pyridine (62 mL)at ambient temperature. To this mixture was added DMTrCl (2.3 g, 6.8mmol). After 1 h, water (2 mL) was added, and it was concentrated invacuo. Ethyl acetate (15 mL), water (5 mL) and brine (1 mL) were added.Layers were separated, and the aqueous layer was extracted with ethylacetate twice (20 mL×2). The combined organics were dried over MgSO₄,concentrated in vacuo and purified by flash column chromatography,eluting with 0 to 80% EtOAc in Hexane to give the desired product. LCMS(ES, m/z): 673.4 [M+H]⁺ 0.1H NMR (500 MHz, DMSO-d₆) δ 12.24 (s, 1H),11.96 (s, 1H), 7.50-7.36 (m, 2H), 7.35-7.10 (m, 9H), 6.96-6.82 (m, 4H),5.88 (d, J=4.1 Hz, 1H), 5.83 (d, J=5.1 Hz, 1H), 5.42 (d, J=5.7 Hz, 1H),4.66 (q, J=4.1 Hz, 1H), 4.45 (td, J=5.9, 3.5 Hz, 1H), 3.81-3.69 (6H),3.65 (ddd, J=8.4, 6.0, 4.3 Hz, 1H), 3.46-3.35 (m, 1H), 3.19 (dd, J=9.4,7.9 Hz, 1H), 2.78 (h, J=6.8 Hz, 1H), 1.22-1.05 (m, 6H).

Preparation 22: ammonium(2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-ylphosphonate

Step 1:N-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)-1,9-dihydro-6H-purin-6-one(1.7 g, 5.7 mmol) was co-evaporated with pyridine (3×5 mL) and then,re-dissolved in pyridine (34 mL). To the mixture at 0° C. was addedchlorotrimethylsilane (4.32 g, 39.8 mmol) dropwise. It was stirred at rtfor 1 h and then, cooled to 0° C. again. Isobutyric anhydride (1.348 g,8.52 mmol) was added dropwise, and it was stirred at rt for 3 h. It wasquenched by the addition of water (8.5 mL). After 5 min, NH₄OH (ca. 29%,17 mL) was added, and the mixture was stirred for 30 min. It wasconcentrated and purified by column chromatography eluted with 1 to 30%MeOH in CH₂Cl₂ to give the product. LCMS (ES, m/z): 396.9 [M+H]⁺. ¹H-NMR(400 MHz, DMSO-d₆): δ 9.52 (br s, 2H), 8.39 (s, 1H), 5.79 (d, J=7.1 Hz,1H), 5.59 (s, 1H), 5.40 (s, 1H), 5.22 (s, 1H), 4.55 (d, J=6.7 Hz, 1H),4.21 (s, 1H), 3.77 (t, J=9.3 Hz, 1H), 3.61 (s, 1H), 3.30 (dt, J=6.4, 3.3Hz, 1H), 2.78 (p, J=6.9 Hz, 1H), 1.13 (d, J=6.8 Hz, 6H).

Step 2:N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dihydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

To a mixture ofN-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-tetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide(480 mg, 1.299 mmol) in pyridine (10 mL) was added4,4′-(chloro(phenyl)methylene)-bis(methoxybenzene) (484 mg, 1.43 mmol).It was stirred at rt for 16 h and then, concentrated. The crude waspurified by column chromatography on silica gel eluted with 1 to 30%MeOH in CH₂Cl₂ (containing 1% Et₃N) to give the product. LCMS (ES, m/z):672.2 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆+D₂O): δ 8.08 (s, 1H), 7.39 (d,J=7.2 Hz, 2H), 7.32 (t, J=7.6 Hz, 2H), 7.26 (dt, J=9.1, 3.3 Hz, 5H),6.94-6.87 (m, 4H), 5.75 (d, J=5.9 Hz, 1H), 4.39 (dd, J=5.9, 3.5 Hz, 1H),4.14 (t, J=3.9 Hz, 1H), 3.74 (s, 6H), 3.49-3.37 (m, 2H), 3.33 (dd,J=14.5, 7.3 Hz, 1H), 2.87-2.67 (m, 1H), 1.11 (dd, J=6.8, 1.6 Hz, 6H).

Step 3:N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramideandN-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)-4-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide

To a solution ofN-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-3,4-dihydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide(580 mg, 0.863 mmol) in DMF (5 mL) at rt was added 1H-imidazole (147 mg,2.16 mmol) and tert-butylchlorodimethylsilane (156 mg, 1.04 mmol). After6 h, the mixture was diluted with EtOAc (50 mL) and washed with sat aqNaHCO₃ (2×20 mL) and brine (20 mL). It was dried (Na₂SO₄), concentrated,and purified by reverse phase (C18) chromatography eluted with 0 to 95%ACN in water to give the products. LCMS (ES, m/z): 786.3 [M+H]⁺.

Step 4.(2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-ylphenyl phosphonate and(2R,3S,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-ylphenyl phosphonate

To a solution of a mixture ofN-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramideand N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)-4-hydroxytetrahydrothiophen-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide(220 mg, 0.280 mmol) in pyridine (2 mL) at 0° C. was added diphenylphosphonate (98 mg, 0.420 mmol). The resulting mixture was stirred at rtfor 20 min. It was used in the next reaction step without purification.LCMS (ES, m/z): 926.2 [M+H]⁺.

Step 5. ammonium(2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-ylphosphonate

To the reaction mixture from Step 4 at 0° C. was added Et₃N (0.28 mL,2.0 mmol) and water (0.28 mL). It was stirred at rt for 30 min. It wasconcentrated, and the residue was partitioned between CH₂Cl₂ (40 mL) andaq NaHCO₃ (5%, 30 mL). The organic layer was washed with aq NaHCO₃ (5%,2×30 mL), dried (Na₂SO₄), concentrated and purified by silica gel columnchromatography using 0-10% MeOH in CHCl₃ containing 1% Et₃N to give amixture. The mixture was further purified by prep-HPLC Prep-HPLC(XBridge Shield RP18 OBD Column, 19×150 mm) eluted with 46 to 79% ACN inaq NH₄HCO₃ (10 mM) over 7 min to give the product. LCMS (ES, m/z): 850.2[M+H]⁺. ¹H-NMR (400 MHz, CD₃OD): δ 8.18 (s, 1H), 7.68 (s, 0.5H),7.59-7.49 (m, 2H), 7.45-7.36 (m, 4H), 7.37-7.30 (m, 2H), 7.28-7.22 (m,1H), 6.95-6.87 (m, 4H), 6.16-6.07 (m, 2H), 4.88-4.87 (m, 1H), 4.69 (dd,J=7.3, 3.3 Hz, 1H), 3.81 (s, 6H), 3.51 (dd, J=4.9, 1.9 Hz, 2H), 3.37 (s,1H), 2.67 (p, J=6.9 Hz, 1H), 1.21 (dd, J=6.9, 0.9 Hz, 6H), 0.77 (s, 9H),0.01 (s, 3H), −0.28 (s, 3H). ³¹P-NMR (162 MHz, DMSO-d₆): δ −0.74 (s,1P).

Preparation 23:2-amino-9-[5-O-(hydroxy{[hydroxy(phosphonooxy)phosphoryl]oxy}-phosphoryl)-β-D-xylofuranosyl]-1,9-dihydro-6H-purin-6-one

Step 1:2-amino-9-[5-O-(hydroxy{[hydroxy(phosphonooxy)phosphoryl]foxy}-phosphoryl)-β-D-xylofuranosyl]-1,9-dihydro-6H-purin-6-one

To a stirred solution of9-(2,3-di-O-acetyl-β-D-xylofuranosyl)-2-[(2-methylpropanoyl)-amino]-1,9-dihydro-6H-purin-6-one(100 mg, 0.229 mmol) in pyridine (0.25 mL) and 1,4-dioxane (0.75 mL) wasadded a freshly prepared solution of2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (50 mg, 0.247 mmol) in1,4-dioxane (0.25 mL). The reaction mixture was stirred at ambienttemperature for 10 min, and then a solution of tributylammoniumpyrophosphate (189 mg, 0.344 mmol) in DMF (0.69 mL) was added, followedby addition of tributylamine (0.23 mL, 0.968 mmol) in one portion atambient temperature. The reaction mixture was stirred for 10 min atambient temperature, and then a solution of iodine (29.0 mg, 0.114 mmol)in pyridine (0.50 mL) and water (0.05 mL) was added. The reactionmixture was stirred for 15 min, excess iodine was quenched with 5% aqNaHSO₃ (3 mL), and the reaction mixture was evaporated to dryness. Theresidue was dissolved in 10 mL H₂O, and after standing at rt for 30 min,28% aq ammonium hydroxide (5 mL) was added. The reaction mixture wasstirred at 50° C. for 5 h. LCMS indicated full conversion to desiredproduct, and the mixture was filtered and lyophilyzed. The product waspurified using mass-directed reverse phase HPLC with a Waters SunFireC18 OBD Prep Column, 100 Å, 5 μm, 19 mm×150 mm, [Waters Part #186002568]using a gradient solvent system with MeCN and 100 mM aq triethylammoniumacetate. Lyophilization of the product fractions furnished2-amino-9-[5-O-(hydroxy{[hydroxy(phosphonooxy)phosphoryl]oxy}phosphoryl)-3-D-xylofuranosyl]-1,9-dihydro-6H-purin-6-oneas the tetra-triethylamine salt. LCMS (ES, m/z): 522 [M−H]⁻.

Preparation 24:((2R,3S,4R,5S)-5-(7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyltetrahydrogen triphosphate

Step 1.((3aR,4R,6S,6aS)-6-(7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-2-methoxytetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methanol

To the stirred suspension of(2S,3R,4S,5R)-2-(7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol(30 mg, 0.105 mmol) in 1,4-dioxane (0.3 mL) was added trimethylorthoformate (0.22 mL, 2.011 mmol) in one portion at ambienttemperature, followed by p-toluenesulfonic acid monohydrate (22 mg,0.116 mmol). The reaction mixture was stirred at ambient temperature for16 h. LCMS indicated significant conversion to desired product, and thecrude mixture was carefully quenched by adding triethylamine (0.05 mL)at 0° C. Following concentration, the residue was purified by flashcolumn chromatography on 12 gram silica gel using a gradient solventsystem with MeOH and CH₂Cl₂. Concentration of the product fractionsfurnished((3aR,4R,6S,6aS)-6-(7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-2-methoxytetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methanol.LCMS (ES, m/z): 310 [M+H]⁺.

Step 2:((3aR,4R,6S,6aS)-6-(7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-2-methoxytetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyltetrahydrogen triphosphate

To the stirred suspension of((3aR,4R,6S,6aS)-6-(7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-2-methoxytetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methanol(16 mg, 0.052 mmol) in pyridine (0.05 mL) and 1,4-dioxane (0.15 mL) wasadded DMF (0.05 mL) to form a homogeneous solution. To this solution wasadded a freshly prepared solution of2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (12 mg, 0.059 mmol) in1,4-dioxane (0.05 mL) at ambient temperature. The reaction mixture wasstirred at ambient temperature for 15 min, and then a solution oftributylammonium pyrophosphate (43 mg, 0.078 mmol) in DMF (0.10 mL) wasadded, followed by tributylamine (0.052 mL, 0.219 mmol). The reactionmixture was stirred at ambient temperature for 15 min, and then asolution of iodine (6.58 mg, 0.026 mmol) in pyridine (0.10 mL) and water(0.01 mL) was added. The reaction mixture was stirred at ambienttemperature for 15 min and excess iodine was quenched with 5% aqueousNaHSO₃ (0.5 mL). LCMS indicated significant conversion to desiredproduct, and the reaction mixture was concentrated to yield ((3aR,4R,6S,6aS)-6-(7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-2-methoxytetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyltetrahydrogen triphosphate, which was used directly in the next reactionstep without additional purification. LCMS (ES, m/z): 548 [M−H]⁻.

Step 3.((2R,3S,4R,5S)-5-(7-amino-H-pyrazolo[4,3-d]pyrimidin-3-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyltetrahydrogen triphosphate

To the stirred solution of crude((3aR,4R,6S,6aS)-6-(7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-2-methoxytetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyltetrahydrogen triphosphate (49.4 mg, 0.090 mmol) in water (0.15 mL) andDMF (0.15 mL) was added formic acid (0.4 mL, 10.60 mmol) in one portion.The reaction mixture was stirred at ambient temperature for 18 h. LCMSindicated significant conversion to desired product, and the mixture wasfiltered and lyophilyzed. The product was purified using mass-directedreverse phase HPLC with a Waters SunFire C18 OBD Prep Column, 100 Å, 5m, 19 mm×150 mm, [Waters Part #186002568] using a gradient solventsystem with MeCN and 100 mM aqueous triethylammonium acetate.Lyophilization of the product fractions furnished ((2R,3 S,4R,5S)-5-(7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyltetrahydrogen triphosphate. LCMS (ES, m/z): 506 [M−H]⁻.

Preparation 25:((2R,3S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyltetrahydrogen triphosphate

Step 1:((2R,3S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyltetrahydrogen triphosphate

To a mixture of 2-fluoroadenosine (200 mg, 0.701 mmol) intrimethylphosphate (1.948 mL, 16.83 mmol) was added tributylamine (0.500mL, 2.104 mmol), and the mixture was stirred 15 min at rt and thencooled in an ice/brine bath. Then POCl₃ (0.137 mL, 1.472 mmol) was addeddropwise with bath temperature maintained at −5 to 0° C. After 1.25 h, a0° C. mixture of tributylammonium pyrophosphate (327 mg, 0.596 mmol),MeCN (2.8 mL) and tributylamine (1.000 mL, 4.21 mmol) were added, andthe mixture was allowed to warm to rt, followed by 16 h stirring at rt.The mixture was purified directly by reverse phase HPLC using a gradientof 1-20% MeCN with 100 mM aqueous triethylammonium acetate to furnish((2R,3S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyltetrahydrogen triphosphate mg. LCMS (ES, m/z): 524 [M−H]⁻.

Preparation 26: 3′-(aminomethyl)-3′-deoxyguanosine 5′-(tetrahydrogentriphosphate)

The title compound was prepared according to published procedures(WO2015161137).

The Preparations below were used as shown or were further modifiedthrough additional synthetic manipulations analogous to those describedin Preparations 1-26.

Preparation 27:2-amino-9-((β-D-xylofuranosyl)-1,9-dihydro-6H-purin-6-one

The title compound was prepared according to published procedures(Journal of Medicinal Chemistry 1987, 30(6), 982-991).

Preparation 28:5-amino-3-((β-D-ribofuranosyl)-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one

The title compound was prepared according to published procedures(Journal of Organic Chemistry 2007, 72(1), 173-179).

Preparation 29:9-{3-azido-5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-deoxy-β-D-ribofuranosyl}-N-(phenylcarbonyl)-9H-purin-6-amine

The title compound was prepared according to published procedures(Bulletin of the Korean Chemical Society 2004, 25(2), 243-248 andNucleosides, Nucleotides & Nucleic Acids 2005 24(10-12), 1707-1727).

Preparation 30: 1-(β-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridin-4-amine

The title compound was prepared according to published procedures(Tetrahedron 1993, 49(3), 557-570).

Preparation 31:1-(β-D-ribofuranosyl)-1,5-dihydro-4H-imidazo[4,5-c]pyridin-4-one

The title compound was prepared according to published procedures(Tetrahedron 1993, 49(3), 557-570)

Preparation 32: 4′-thioadenosine

The title compound was prepared according to published procedures(Journal of Medicinal Chemistry 2006, 49(5), 1624-1634).

Preparation 33:7-((β-D-ribofuranosyl)-3,7-dihydro-4H-imidazo[4,5-d][1,2,3]triazin-4-one

The title compound was prepared according to published procedures(Organic & Biomolecular Chemistry 2014, 12(23), 3813-3815).

Preparation 34: 3-β-D-ribofuranosyl)-3H-imidazo[4,5-b]pyridin-7-amine

The title compound was prepared according to published procedures(Biochemistry 2005, 44(37), 12445-12453).

Preparation 35:7-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

The title compound was prepared according to published procedures(Journal of the Chemical Society, Perkin Transactions 1: Organic andBio-Organic Chemistry 1995 (12), 1543-50).

Preparation 36:1-(3-deoxy-β-D-erythro-pentofuranosyl)-1,5-dihydro-4H-imidazo[4,5-c]pyridin-4-one

The title compound was prepared according to published procedures(Chemical & Pharmaceutical Bulletin 1996, 44(2), 288-295).

Preparation 37:7-(2-deoxy-β-D-erythro-pentofuranosyl)-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-4-amine

The title compound was prepared according to published procedures(Synthesis 2006 (12), 2005-2012).

Preparation 38:(2S,3R,4S,5R)-2-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol

The title compound was prepared according to published procedures(WO2015148746).

Preparation 39:N-(8-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)pyrazolo[1,5-a][1,3,5]triazin-4-yl)benzamide

The title compound was prepared according to published procedures(WO2015148746).

Preparation 40:7-(2-deoxy-4-ethynyl-β-D-erythro-pentofuranosyl)-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-4-amine

The title compound was prepared according to published procedures(WO2015148746).

Preparation 41:9-(2-chloro-2-deoxy-β-D-arabinofuranosyl)-9H-purin-6-amine

The title compound was prepared according to published procedures(Journal of the American Chemical Society 1996, 118(46), 11341-11348).

Preparation 42: 2′-deoxy-2′-methyladenosine

The title compound was prepared according to published procedures(Synthesis 2005 (17), 2865-2870).

Preparation 43:(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((R)-1-hydroxyethyl)tetrahydrofuran-3,4-diol

The title compound was prepared according to published procedures(Bioorganicheskaya Khimiya 1989, 15(7), 969-975).

Preparation 44:5-fluoro-7-(β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

The title compound was prepared according to published procedures(Nucleosides, Nucleotides, & Nucleic Acids 2004, 23(1-2), 161-170).

Preparation 45:1-(β-D-ribofuranosyl)-1,5-dihydro-4H-imidazo[4,5-d]pyridazin-4-one

The title compound was prepared according to published procedures(Journal of the Chemical Society, Perkin Transactions 1: Organic andBio-Organic Chemistry (1972-1999), 1989 (10), 1769-1774).

Preparation 46:2-amino-9-[(2R,3R,4S,5S)-5-fluoro-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-1,9-dihydro-6H-purin-6-one

The title compound was prepared according to published procedures(WO2014099941)

Preparation 47:2-amino-9-((2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)-4-methylenetetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one

The title compound was prepared according to published procedures(Journal of Medicinal Chemistry 1992, 35, 2283-2293).

Preparation 48:(2S,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol

The title compound was prepared according to published procedures(Tetrahedron Letters 1994, 35(30), 5339).

The following experimental procedures detail the preparation of specificexamples of the instant disclosure. The compounds of the examples aredrawn in their neutral forms in the procedures and tables below. In somecases, the compounds were isolated as salts depending on the method usedfor their final purification and/or intrinsic molecular properties. Theexamples are for illustrative purposes only and are not intended tolimit the scope of the instant disclosure in any way.

EXAMPLES Example 1:(5R,7R,8R,12aR,14R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-16-hydroxyoctahydro-12H-5,8-methanofuro[3,2-1][11,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate2,10-dioxide

Step 1:(2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl(2-cyanoethyl) phosphonate

To a solution of(2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite (3 g, 3.09 mmol) in ACN (15mL) was added water (0.111 mL, 6.18 mmol) and pyridin-1-ium2,2,2-trifluoroacetate (0.717, 3.71 mmol). The resulting mixture wasstirred at RT, and the reaction progress was monitored by LCMS/TLC.After the phosphoramidite was consumed, the reaction mixture containingthe product was used in the next step without purification. LCMS (ES,m/z): 887.4 [M+H]⁺.

Step 2: 2-methylpropan-2-aminium(2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To the reaction mixture from Step 1 (assumed to contain 3.09 mmol of(2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(tert-butyldimethylsilyloxy)-2-(2-isobutyramido-6-oxo-1,6-dihydropurin-9-yl)-tetrahydrofuran-3-yl2-cyanoethyl phosphonate) was added tert-butylamine (15.0 mL, 142 mmol)in one portion, and the resulting solution was stirred at rt for 40 min.It was concentrated, and the residue was co-evaporated with ACN (2×15mL) to give the product, which was used for the next step withoutfurther purification. LCMS (ES, m/z): 832.3 [M−H]⁻.

Step 3: pyridin-1-ium(2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To a stirred solution of crude 2-methylpropan-2-aminium(2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate (˜4.2 g, ˜3.09 mmol, from step 2) in CH₂Cl₂ (37 mL) wereadded water (0.558 mL, 31.0 mmol) and dichloroacetic acid in CH₂Cl₂ (6%,37 mL, 31.5 mmol). It was stirred for 40 min. Then, triethylsilane (60mL) was added, and the solution was stirred for 1.5 h. Pyridine (4.5 mL)was added to the reaction. It was concentrated. The residue wastriturated with MTBE (50 ml) and Hexane (50 mL), and the supernatant wasdecanted. This process was repeated twice. The crude mixture was keptover P₂O₅ under reduced pressure for 20 h to give a crude mixturecontaining the product. LCMS (ES, m/z): 532.2 [M+H]⁺.

Step 4.(2R,3R,4R,5R)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphanyl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To a stirred solution of pyridin-1-ium(2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate (680 mg, crude, ˜0.722 mmol) in ACN (5 mL) under Ar wasadded activated 4 Å molecular sieves (100 mg). The resulting mixture wasstirred at rt over 30 min.(2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (0.805 g,0.939 mmol) was co-evaporated with ACN (3×1 mL), re-dissolved in ACN (5mL), and dried by adding activated 4 Å molecular sieve (100 mg). After30 min, it was added to the previously prepared mixture containingpyridin-1-ium(2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate. The mixture was stirred at rt for 1 h. The reaction mixturecontaining the product was used in the next reaction step immediatelywithout purification. LCMS (ES, m/z): 1288.4 [M+H]⁺.

Step 5.(2R,3R,4R,5R)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To the reaction mixture containing the crude(2R,3R,4R,5R)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphanyl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate (˜1 mmol, with excess pyridinium 2,2-dichloroacetate) wasadded tert-butyl hydroperoxide in decane (5.5M, 0.64 mL, 3.5 mmol)dropwise. It was stirred at rt for 1 h. Then, the solution was cooled to0° C., and NaHSO₃ (250 mg) in water (5 mL) was added slowly. After 5min, the mixture was concentrated, and the residue was purified byreverse phase (C18) chromatography eluted with 5 to 45% ACN in aqNH₄HCO₃ (0.04%) to give the product. LCMS (ES, m/z): 1305.6 [M+H]⁺.

Step 6.(2R,3R,4R,5R)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To a stirred solution of(2R,3R,4R,5R)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate (340 mg, 0.239 mmol) in CH₂Cl₂ (4 mL) were added water (44.5mg, 2.468 mmol) and dichloroacetic acid (0.280 g, 2.17 mmol) in CH₂Cl (5ml). It was stirred at rt for 30 min. Et₃SiH (4 mL) was then added, andthe mixture was stirred for 1.5 h. Pyridine (3 mL) was added to thereaction, and it was concentrated to give a crude product, which wasused for the next step without purification. LCMS (ES, m/z): 1002.4[M+H]⁺.

Step 7:(5R,7R,8R,12aR,14R,15aS,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate2-oxide

Crude(2R,3R,4R,5R)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate (1.5 g, ˜0.24 mmol) was co-evaporated with pyridine (3×5 mL)and then re-dissolved in pyridine (4 mL). To the reaction was added2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (160 mg, 0.865mmol) in one portion. The resulting mixture was stirred at rt for 1 h.It was used for the next reaction step directly without purification.LCMS (ES, m/z): 984.3 [M+H]⁺.

Step 8.(5R,7R,8R,12aR,14R,15aS,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate2,10-dioxide

To the stirred mixture containing(5R,7R,8R,12aR,14R,15aS,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate2-oxide was added water (156 mg, 8.65 mmol) and iodine (81 mg, 0.321mmol). After 10 min, the mixture was poured into a solution of NaHSO₃(52 mg) in water (36 ml), and it was stirred for 5 min. It was cooled to0° C., and NaHCO₃ (1.04 g) was slowly added. After 5 min, EtOAc (50 mL)and Et₂O (50 ml) were added. Layers were separated, and the aq layer wasextracted with EtOAc (1×30 ml). The organic layers were combined,concentrated, and purified by silica gel column chromatography elutedwith 0-20% MeOH in CH₂Cl₂ to give the product. LCMS (ES, m/z): 998.3[M+H]⁺.

Step 9:(5R,7R,8R,12aR,14R,15aS,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino-9H-purin-9-yl}octahydro-12H-5,8-methanofuro3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate2,10-dioxide

To a stirred solution of(5R,7R,8R,12aR,14R,15aS,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate2,10-dioxide (160 mg) in ACN (2 mL) was added tert-butylamine (2 mL) atrt. After 30 min, it was concentrated to give the crude product, whichwas used for the next step without purification. LCMS (ES, m/z): 945.2[M+H]⁺.

Step 10:(5R,7R,8R,12aR,14R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate2,10-dioxide

Crude(5R,7R,8R,12aR,14R,15aS,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate2,10-dioxide (220 mg) was dissolved in a solution of MeNH₂ in EtOH (30%,4 mL), and it was stirred at rt for 5 h. Then, the volatile componentwas removed under reduced pressure to give a crude product, which wasused for the next reaction step without purification. LCMS (ES, m/z):773.2 [M+H]⁺ and 771.3 [M−H]⁻.

Step 11:(5R,7R,8R,12aR,14R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-16-hydroxyoctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate2,10-dioxide

The crude from Step 10 was co-evaporated with pyridine (2.5 ml) and Et₃N(2.5 mL) three times. It was dissolved in pyridine (2 mL). To thesolution was added Et₃N (1.51 g, 14.9 mmol) and triethylaminetrihydrofluoride (1.2 g, 7.45 mmol) dropwise. The mixture was heated at50° C. for 5 h. Then, it was concentrated and purified bypreparative-HPLC (T3 Prep Column, 100 Å, 5 μm, 19 mm×250 mm) eluted with0 to 10% ACN in aq NH₄HCO₃ (50 mM) to give the product. LCMS (ES, m/z):657.1 [M−H]⁻. ¹H-NMR: (300 MHz, DMSO-d₆+D₂O): δ 8.35 (s, 1H), 8.16 (s,1H), 8.03 (s, 1H), 6.34 (dd, J=8.8, 5.9 Hz, 1H), 5.85 (d, J=8.3 Hz, 1H),5.12-4.98 (m, 2H), 4.36 (d, J=3.9 Hz, 1H), 4.22 (t, J=7.2 Hz, 1H), 4.09(s, 1H), 3.96-3.79 (m, 4H), 3.09-2.99 (m, 1H), 2.64-2.51 (m, 1H).³¹P-NMR: (121 MHz, DMSO-d₆+D₂O): δ −1.65 (s), −2.36 (s).

Examples 2 through 19, shown in Table 1 below, were prepared accordingto procedures analogous to those outlined in Example 1 above using theappropriate nucleoside monomers, described as Preparations or asobtained from commercial sources.

TABLE 1 Mass Ex. Structure Name [M − H]⁻  2

(5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-15-fluoro-16-hydroxyoctahydro-12H-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine- 2,10-diolate 2,10-dioxide 675  3

(5R,7R,8R,12aR,14R,15aS,16R)-7,14-bis(6-amino-9H-purin-9-yl)-16-hydroxyoctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecine- 2,10-diolate2,10-dioxide 657  4

(5R,7R,8R,12aR,14R,15R,15aR,16R)-7,14-bis(6-amino-9H-purin-9-yl)-15-fluoro-16-hydroxyoctahydro-12H-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine- 2,10-diolate 2,10-dioxide 659  5

(5R,7R,8S,12aR,14R,15R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-16-fluoro-15-hydroxyoctahydro-12H-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine- 2,10-diolate 2,10-dioxide 675  6

(5R,7R,8R,12aR,14R,15R,15aS,18R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-18-hydroxyhexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine- 2,10(12H)-diolate 2,10-dioxide 685  7

(5R,7R,8R,12aR,14R,15S,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-15-fluoro-16-hydroxyoctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate 2,10-dioxide 675  8

(5R,7R,8R,12aR,14R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-16-hydroxy-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate 2,10-dioxide 660 [M + H]⁺ 9

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15,16-dihydroxyoctahydro-12H-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine- 2,10-diolate 2,10-dioxide 672 10

(5R,7R,8R,12aR,14R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-hydroxyoctahydro-12H-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine- 2,10-diolate 2,10-dioxide 656 11

(5R,7R,8R,12aR,14R,15R,15aS,16S)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-15,16-dihydroxyoctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecine-2,10- diolate 2,10-dioxide 689 12

(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-12a-ethynyl-15,16-dihydroxyoctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine- 2,10-diolate 2,10-dioxide 699 [M +H]⁺ 13

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-2,10,15-trihydroxy-16-methoxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 68714

2-amino-9- [(5S,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-azido-2,10,15-trihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 69815

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-chloro-2,10,16-trihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 69116

2-amino-9- [(5S,7R,8R,12R,12aR,14R,15R,15aS)-14-(6-amino-9H-purin-9-yl)-2,10,15-trihydroxy-12-methyl-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 67117

2-amino-9- [(2aR,5S,6aS,7R,8R,9aR,12S,14R,14aS,15R)-8-(6-amino-9H-purin-9-yl)-5,7,12-trihydroxy-5,12-dioxidohexahydro-6aH-2a,14- (epoxymethano)furo[3,2-d]oxeto[2,3-k][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-15(2H,3H)-yl]-1,9-dihydro-6H-purin-6-one 685 18

2-amino-9-[(5S,7R,8R,12S,12aS,14R,15R,15aS)-14-(6-amino-9H-purin-9-yl)-2,10,15-trihydroxy-12-methyl-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 67119

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aS)-14-(6-amino-9H-purin-9-yl)-2,10-dihydroxy-15-methyl-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 655

Example 20:(5R,7R,8S,12aR,14R,15R,15aS,18R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-18-fluorohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10(12H-diolate2,10-dioxide

Step 1.(2R,3514R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphenyl phosphonate

To a stirred solution of N-(9-((2R,3 S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide(1 g, 1.520 mmol) in pyridine (7.6 mL) under Ar was added diphenylphosphonate (1.068 g, 4.56 mmol), and it was stirred at rt for 20 min.The reaction mixture containing the product was used for the nextreaction step without purification.

Step 2. (2R,3,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To the reaction mixture from Step 1 was added water (1.5 ml) and Et₃N(1.5 mL). The mixture was stirred at rt for 20 min. Then, it wasconcentrated, and the residue was partitioned between CH₂Cl₂ (50 mL) andaq NaHCO₃ (5%, 20 mL). Layers were separated. The organic layer waswashed with aq NaHCO₃ (5%, 2×20 mL), dried (Na₂SO₄), concentrated andpurified by silica gel column chromatography using 0 to 7% MeOH inCH₂Cl₂ (1% Et₃N) to give the product. LCMS (ES, m/z): 722 [M+H]⁺.³¹P-NMR: (162 MHz, CD₃OD): δ 2.73 (s, 1P).

Step 3.(2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To a stirred solution of the product of Step 2 (0.9 g, 0.999 mmol) inCH₂Cl₂ (6 mL) were added water (0.180 g, 9.99 mmol) and2,2-dichloroacetic acid (1.16 g, 8.99 mmol) in CH₂Cl₂ (10 ml). Themixture was stirred at rt for 15 min. Et₃SiH (10 mL) was added, and itwas stirred for 1 h. Then, pyridine (2 mL) was added, and it wasconcentrated. The residue was purified by reverse phase (C18)chromatography eluted with 0 to 30% ACN in aq NH₄HCO₃ (0.04%) to givethe product. LCMS (ES, m/z): 722 [M−H]⁻. 417.9. ¹H-NMR: (400 MHz,CD₃OD): δ 8.31 (s, 1H), 7.49 (d, J=1.6 Hz, 0.5H), 6.15 (d, J=6.8 Hz,1H), 5.91 (d, J=1.6 Hz, 0.5H), 5.42-5.32 (m, 1.5H), 5.21 (dd, J=4.5, 1.9Hz, 0.5H), 4.45-4.32 (m, 1H), 3.81 (d, J=3.5 Hz, 2H), 3.20 (q, J=7.4 Hz,1H), 2.73 (p, J=6.9 Hz, 1H), 1.30 (t, J=7.3 Hz, 1.5H), 1.23 (d, J=6.9Hz, 6H). ¹⁹F-NMR: (376 MHz, CD₃OD): δ −200.96 (s, 1F). ³¹P-NMR: (162MHz, CD₃OD): δ 2.41 (s, 1P).

Step 4:(2R,3S,4R,5R)-5-((((((1R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphanyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

(2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate (600 mg, 0.676 mmol) was co-evaporated with ACN (3×5 mL),re-dissolved in ACN (3 mL), dried by addition of activated 4 Å molecularsieves (150 mg) and kept under Ar.(1R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl(2-cyanoethyl) diisopropylphosphoramidite (available from Exiqon(EQ-0063-1000), 235.5 mg, 0.56 mmol) and pyridinium2,2,2-trifluoroacetate (162 mg, 0.84 mmol) were co-evaporated with ACN(3×5 mL), re-dissolved in ACN (5 mL), and dried by addition activated 4Å molecular sieve (150 mg). After 30 min, it was added to the previouslyprepared mixture containing(2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate. The mixture was stirred at rt for 1 h. The reaction mixturecontaining the product was used in the next reaction step immediatelywithout purification. LCMS (ES, m/z): 1202.3 [M−H]⁻.

Step 5. (2R,3S,4R,5R)-5-((((((R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To the stirred reaction mixture from Step 4 was added tert-butylhydroperoxide in decane (5.5M, 0.31 mL, 1.71 mmol) dropwise. Theresulting mixture was stirred at rt for 1 h. After 30 min, the solutionwas cooled to 0° C., and NaHSO₃ (150 mg) in water (5 mL) was addedslowly. After 5 min, the mixture was concentrated, and the residue waspurified by reverse phase (C18) chromatography eluted with 0 to 75% ACNin aq NH₄HCO₃ (5 mM) to give the product. LCMS (ES, m/z): 1220.1 [M+H]⁺.¹⁹F-NMR (376 MHz, CD₃OD): δ −200.38, −202.45 (2s, 1F). ³¹P-NMR: (162MHz, CD₃OD): δ 2.57, 2.49 (2s, 1P); −3.52, −4.21 (2s, 1P).

Step 6. (2R,3S,4R,5R)-5-((((((S,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To a solution of(2R,3S,4R,5R)-5-((((((1R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate (190 mg, 0.16 mmol) in CH₂Cl₂ (2.5 mL) was added water (28.8mg, 1.6 mmol) and dichloroacetic acid in CH₂Cl₂ (0.6M, 2.5 mL). Themixture was stirred at rt for 10 min, and then Et₃SiH (4.5 mL) wasadded. After 1 h, pyridine (0.5 mL) was added. After 10 min, theresulting mixture was concentrated to give the product, which was usedfor the next reaction step without purification. LCMS (ES, m/z): 917.9[M+H]⁺. ³¹P-NMR: (162 MHz, CD₃OD): δ 2.51, 2.34 (2s, 1P); −3.46, −3.82(2s, 1P).

Step 7:(5R,7R,8S,12aR,14R,15R,15aS,18R)-2-(2-cyanoethoxy)-18-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10(12H)-olate 2,10-dioxide

To pyridine (16 mL) under Ar was added diphenyl chlorophosphate (0.66mL, 3.2 mmol). It was cooled at −40° C. and then, a solution of crudefrom Step 6 in CH₂Cl₂ (16 mL) was added dropwise over 20 min. Theresulting mixture was stirred at −40° C. for 40 min. The reactionmixture was used in the next step without purification. LCMS (ES, m/z):898.2 [M−H]⁻.

Step 8.(5R,7R,8S,12aR,14R,15R,15aS,18R)-2-(2-cyanoethoxy)-18-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10(12H)-olate2,10-dioxide[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10(12H)-diolate 2,10-dioxide

To the solution from Step 7 at 0° C. was added I₂ in pyridine/water(9/1) (3%, 1.76 mL) over 5 min. The mixture was stirred at rt for 40min. Then, it was treated with a solution of Na₂S₂O₃.5H₂O (150 mg) inwater (2 mL). After 5 min, the mixture was concentrated under reducedpressure. The residue was purified by reverse phase (C18) chromatographyeluted with 0 to 45% ACN in aq NH₄HCO₃ (0.04%) to give the product. LCMS(ES, m/z): 915.8 [M+H]⁺. ¹⁹F-NMR (376 MHz, CD₃OD): δ −198.70, −203.36(2s, 1F). ³¹P-NMR (162 MHz, CD₃OD): δ −0.96, −1.75 (2s, 1P); −3.64,−4.71 (2s, 1P).

Step 9:(5R,7R,8S,12aR,14R,15R,15aS,18R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-18-fluorohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10(12H)-diolate2,10-dioxide

(5R,7R,8S,12aR,14R,15R,15aS,18R)-2-(2-cyanoethoxy)-18-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10(12H)-olate2,10-dioxide (110 mg, 0.12 mmol) was dissolved in a solution of MeNH₂ inEtOH (30%, 15 mL), and the resulting solution was stirred at rt for 3 h.Then, it was concentrated, and the residue was purified bypreparative-HPLC (Atlantis Prep T3 Column, 19×250 mm) eluted with 0 to9% ACN in aq NH₄HCO₃ (50 mM) to give the product. LCMS (ES, m/z): 686.9[M−H]⁻. ¹H-NMR (400 MHz, D₂O): δ 8.14 (s, 1H), 7.85 (s, 1H), 7.80 (s,1H), 6.01 (s, 1H), 5.99 (d, J=8.5 Hz, 1H), 5.84-5.66 (m, 1H), 5.44 (d,J=3.6 Hz, 0.5H), 5.31 (d, J=3.6 Hz, 0.5H), 4.96 (d, J=3.9 Hz, 1H), 4.84(s, 1H), 4.65-4.53 (m, 1H), 4.33-4.15 (m, 4H), 4.10 (d, J=8.2 Hz, 1H),3.96 (d, J=8.2 Hz, 1H). ¹⁹F-NMR (376 MHz, D₂O): δ −199.02 (s, 1F).³¹P-NMR (162 MHz, D₂O): δ −1.89 (s, 1P), −2.49 (s, 1P).

Examples 21 through 29, as shown in Table 2 below, were preparedaccording to procedures analogous to those outlined in Example 20 aboveusing the appropriate monomers, described as Preparations or as obtainedfrom commercial sources, in the coupling step.

TABLE 2 Mass Ex. Structure Name [M − H] 21

2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aR)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10-dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 65922

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-fluoro-2,10-dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 65923

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 67724

2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(6-amino-9H-purin-9-yl)-2,10-dihydroxy-2,10-oxidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7(12H)-yl]- 1,9-dihydro-6H-purin-6-one669 25

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10-dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 65926

2-amino-9- [(2S,5R,7R,8S,10S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 67727

2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(6-amino-9H-purin-9-yl)-2,10-dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 64128

2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(6-amino-9H-purin-9-yl)-2,10-dihydroxy-15-methyl-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 65529

2-amino-9-[(5S,8R,12aR,15S,15aR)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10-dihydroxy-15-methyl-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one 673

Example 30:2-amino-7-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one

cGAS buffer consisted of 40 mM Tris-HCL, pH 7.5, 100 uM NaCl, 10 mMMgCl₂. cGAS enzyme was purchased from Novoprotein (Novoprotein code:SGCAS), having been expressed in E. coli and purified using a HIS tag.The calculated molecular weight was 55.3 kDa, and the sequence was:

(SEQ. ID. NO. 1) MAHHHHHHGSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLTFLYDGIEIQADQTPEDLDMEDNDIIEAHREQIGGENLYFQGGASKLRAVLEKLKLSRDDISTAAGMVKGVVDHLLLRLKCDSAFRGVGLLNTGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKIIKEEINDIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRKQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCENKEEKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNLFSSNLIDKRSKEFLTKQIEYERNNEFPVFDEF

To a vial were added Herring DNA (CAS #9007-49-2, 0.3 mg/mL in cGASbuffer; 14.8 mL) and cGAS enzyme (3.1 mg/mL in cGAS buffer; 0.78 mL),and the mixture was incubated at RT for 15 min. 7-Deaza-GTP (TriLinkcatalog # N-1044; 5 mM in cGAS buffer, 1.95 mL, 9.75 μmol) and ATP (5 mMin cGAS buffer, 1.95 mL, 9.75 μmol) were added, and the mixture wasincubated on a Radleys Metz heater shaker set to maintain 37° C. whileshaking at 250 rpm for 16 h, after which the mixture was filtered andlyopholyzed. The product was purified using mass-directed reverse phaseHPLC with a Waters SunFire C18 OBD Prep Column, 100 Å, 5 μm, 19 mm×150mm, [Waters Part #186002568] using a gradient solvent system with MeCNand 100 mM aqueous triethylammonium acetate. Lyopholization of theproduct fractions furnished the title compound. LCMS (ES, m/z): 672[M−H]⁻. ¹H NMR (600 MHz, DMSO-d₆) δ 10.33 (s, 1H), 8.37 (s, 1H), 8.10(s, 1H), 7.56 (s, 1H), 7.24 (s, 2H), 6.94 (d, J=3.4 Hz, 1H), 6.28-6.24(m, 3H), 6.01 (d, J=8.1 Hz, 1H), 5.86 (d, J=7.9 Hz, 1H), 5.61 (s, 1H),5.01-4.97 (m, 1H), 4.86-4.83 (m, 1H), 4.70 (s, 1H), 4.25 (s, 1H), 4.21(dd, J=10.4, 4.8 Hz, 1H), 4.03-3.92 (4, 3H), 3.80-3.75 (m, 1H), 3.69 (d,J=12.1 Hz, 1H), 2.76 (s, 12H), 1.02 (S, 18H).

Examples 31 to 65 in Table 3 below were made using procedures analogousto those described above for Example 30 using the appropriate nucleosidetriphosphate monomers. Where necessary, the triphosphates were formedaccording to methods similar to those described for Preparations 23 to26 or by submission of the requisite 5′-OH nucleoside monomer toNuBlocks LLC (Oceanside, Calif.). Example 38 was made using ATP andα-thio-GTP (BIOLOG Life Science Institute, catalog # G014/G015).

TABLE 3 Mass Ex. Structure Name [M − H]⁻ 31

2-amino-9- {(5R,7R,8R,12aR,14R,15R,15aS,16R)-2,10,15,16-tetrahydroxy-14-[6- (methyl amino)-9H-purin-9-yl]-2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl}- 1,9-dihydro-6H-purin-6-one 68732

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-chloro-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 69233

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 69034

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-2-fluoro-9H-purin-9-yl)- 2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 69135

9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 65736

2-amino-9- [(5S,7R,8R,12aR,14R,15R,15aS,16R)-16-(aminomethyl)-14-(6-amino-9H-purin-9- yl)-2,10,15-trihydroxy-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 68637

2-amino-7- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)- 2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4- one 671 38

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-pufin-9-yl)-2,10,15,16-tetrahydroxy-10-oxido-2-sulfidooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one 689 39

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16S)-14-(6-amino-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 67340

2-amino-9- [(5R,7R,8R,12aR,14S,15S,15aS,16R)-14-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 67341

2-amino-9- [(5R,7R,8R,12aR,14S,15S,15aS,16R)-14-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 67242

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(2,6-diamino-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 68843

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-10,15,16-trihydroxy-2,10-dioxido-2- sulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 68844

9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 3,9-dihydro-1H-purine-2,6-dione674 45

2-amino-9- [(5S,7R,8R,12aR,14R,15R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10,15-trihydroxy-2,10-dioxidooctahydro- 12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one 656 46

2-amino-9- [(5S,7R,8R,12aR,14R,15R,15aS,16S)-14-(6-amino-9H-purin-9-yl)-5-fluoro- 2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 69147

9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(2,6-diamino-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 67348

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aS,16R)-14-(6-amino-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 67349

2-amino-9- [(5R,7R,8R,12aR,14S,15S,15aS,16R)-14-(7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 67350

9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(6-amino-9H-purin-9-yl)-2,10,15-trihydroxy- 2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 64251

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(4-amino-1H-imidazo[4,5-c]pyridin-1-yl)- 2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 67252

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aS,16S)-14-(6-amino-9H-purin-9-yl)-16-fluoro-2,10,15-trihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 67553

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purine-6-thione689 54

2-amino-9- [(5R,7R,8R,12aR,14S,15S,15aS,16R)-14-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 67355

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 67456

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-ethyl-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 68657

2-amino-9- {(5R,7R,8R,12aR,14R,15R,15aS,16R)-2,10,15,16-tetrahydroxy-14-[6-(2- methoxyethyl)-9H-purin-9-yl]-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl}- 1,9-dihydro-6H-purin-6-one 71658

1-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,5-dihydro-4H-imidazo[4,5-c]pyridin-4- one 657 59

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(2-amino-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 67360

4-amino-7- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)- 2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile 697 61

1-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,5-dihydro-4H-imidazo[4,5-c]pyridin-4- one 656 62

2-amino-9- [(5S,7R,8R,12aR,14R,15R,15aS)-14-(6-amino-9H-purin-9-yl)-2,10,15-trihydroxy-16-methylidene-2,10-dioxidooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]/ pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one 669 63

7-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]- 3,7-dihydro-4H-imidazo[4,5-d][1,2,3]triazin-4-one 659 64

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(7-amino-3H-imidazo[4,5-b]pyridin-3-yl)- 2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 67265

1-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,5-dihydro-4H-imidazo[4,5-d]pyridazin-4- one 658

Example 66:1-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

cGAS buffer consisted of 40 mM Tris-HCL, pH 7.5, 100 uM NaCl, 10 mMMgCl₂. cGAS enzyme was purchased from Novoprotein (Novoprotein code:SGCAS), having been expressed in E. coli and purified using a HIS tag.The calculated molecular weight was 55.3 kDa, and the sequence was:

(SEQ. ID. NO. 1) MAHHHHHHGSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLTFLYDGIEIQADQTPEDLDMEDNDIIEAHREQIGGENLYFQGGASKLRAVLEKLKLSRDDISTAAGMVKGVVDHLLLRLKCDSAFRGVGLLNTGSYYEHVKISAPNEFDVMFKLEVPRIQLEEYSNTRAYYFVKFKRNPKENPLSQFLEGEILSASKMLSKFRKIIKEEINDIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPASTQEGLRIQNWLSAKVRKQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGKSKTCCENKEEKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHVCTQNPQDSQWDRKDLGLCFDNCVTYFLQCLRTEKLENYFIPEFNLFSSNLIDKRSKEFLTKQIEYERNNEFPVFDEF

To a vial were added Herring DNA (CAS #9007-49-2, 0.3 mg/mL in cGASbuffer; 15.2 mL) and cGAS enzyme (3.1 mg/mL in cGAS buffer; 0.8 mL), andthe mixture was incubated at RT for 15 min. ATP (5 mM in cGAS buffer,2.0 mL, 10 μmol), 7-deaza-8-aza-ITP (5 mM in cGAS buffer, 2.0 mL, 10μmol), and DMSO (5 mL) were added, and the mixture was incubated on aRadleys Metz heater shaker set to maintain 37° C. while shaking at 250rpm for 3d. The mixture was filtered, lyopholyzed, and purified byreverse phase HPLC (eluting acetonitrile/water gradient with 100 mM TEAAmodifier, linear gradient) to afford the title compound as the TEA salt.LCMS (ES, m/z): 658 [M−H]⁻. ¹H NMR (600 MHz, D₂O): δ 8.36 (s, 1H), 8.34(s, 1H), 8.13 (s, 1H), 7.51 (s, 1H), 6.41 (d, J=8.2 Hz, 1H), 6.24 (s,1H), 5.69 (m, 1H), 5.42 (m, 1H), 4.88 (d, J=4.4 Hz, 1H), 4.66 (d, J=4.1Hz, 1H), 4.51 (m, 1H), 4.43 (m, 2H), 4.23 (m, 2H), 4.01 (m, 1H).

Examples 67 to 74 in Table 4 below were made using procedures analogousto those described above for Example 66 using the appropriate nucleosidetriphosphate monomers.

Where necessary, the triphosphates were formed according to methodssimilar to those described for Preparations 23 to 26 or by submission ofthe requisite 5′-OH nucleoside monomer to NuBlocks LLC (Oceanside,Calif.).

TABLE 4 Mass Ex Structure Name [M − H]⁻ 67

9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10,15-trihydroxy-2,10-dioxidooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one 641 68

7-[(5R,7S,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]imidazo[2,1-f][1,2,4]triazin-4(3H)-one 658 69

5-amino-3- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one 674 70

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-2,10,15,16-tetrahydroxy-14-(6-methyl-9H-purin-9-yl)-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 67271

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 69172

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-2,10,15,16-tetrahydroxy-2,10-dioxido-14-(9H-purin-9-yl)octahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 65873

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(4-amino-1H-[1,2,3]triazolo[4,5-c]pyridin-1-yl)-2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 67374

3-[(5R,7S,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7- one 658

Example 75:2-Amino-9-[(5S,7R,8R,12aR,14R,15R,15aS,16R)-16-amino-14-(6-amino-9H-purin-9-yl)-2,10,15-trihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][11,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

Step 1:2-Amino-9-[(5S,7R,8R,12aR,14R,15R,15aS,16R)-16-amino-14-(6-amino-9H-purin-9-yl)-2,10,15-trihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

To a stirred solution of2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-azido-2,10,15-trihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Example 14, 4.0 mg, 0.0055 mmol) in absolute EtOH (1.0 mL) anddeionized water (1.0 mL) was added palladium on carbon (1.0 mg, 10 wt. %loading) in one portion under Ar at RT. The reaction vessel was thenflushed with hydrogen gas and attached to a hydrogen gas balloon. Thereaction mixture was left to stir for 48 h, filtered, and concentratedto afford the title compound. LCMS (ES, m/z): 672 [M−H]⁻. (600 MHz,DMSO-d₆) δ 10.70 (s, 1H), 8.42 (s, 1H), 8.12 (s, 1H), 7.96 (s, 1H), 7.70(s, 1H), 7.29 (br, 2H), 6.56 (br, 2H), 6.00 (d, J=8.3 Hz, 1H), 5.89 (d,J=8.5 Hz, 1H), 5.21 (s, 1H), 5.04 (t, J=6.0 Hz, 1H), 4.16 (s, 1H), 4.05(dd, J=10.5, 5.0 Hz, 1H), 4.00 (s, 1H), 3.77 (d, J=4.1 Hz, 1H), 3.67 (m,2H). ³¹P NMR: (202 MHz, DMSO-d₆): δ −0.4 (s), 2.0 (s).

Alternatively, Example 75 may be prepared from the requisite monomers,according to a method similar to that described above for Example 30.

Example 76:9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purine-6-carboxamide

To a stirred solution of2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-chloro-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one,(Example 32, 16 mg, 0.018 mmol) in DMSO (1.7 mL) was added sodiumcyanide (8.0 mg, 0.16 mmol) in one portion under Ar at RT. The reactionmixture was heated to 80° C. and left to stir at the same temperaturefor 3 h, cooled to ambient temperature, then quenched with cold aceticacid (15 uL). The mixture was filtered and lyophilyzed. The product waspurified using mass-directed reverse phase HPLC with a Waters SunFireC18 OBD Prep Column, 100 Å, 5 μm, 19 mm×150 mm, [Waters Part #186002568]using a gradient solvent system with MeCN and 100 mM aqueoustriethylammonium acetate. Lyophilization of the product fractionsfurnished9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purine-6-carbonitrile.LCMS (ES, m/z): 683 [M−H]⁻.

To a stirred suspension of9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purine-6-carbonitrile(3.0 mg, 0.003 mmol) in deionized water (338 uL) was addedHydrido(dimethylphosphinous acid-kP)[hydrogenbis(dimethylphosphinito-kP)]platinum(II) (1.0 mg, 0.002 mmol). Thereaction mixture was heated to 85° C. and left to stir at the sametemperature for 6 h, cooled to ambient temperature, filtered andlyopholyzed. The product was purified using mass-directed reverse phaseHPLC with a Waters SunFire C18 OBD Prep Column, 100 Å, 5 μm, 19 mm×150mm, [Waters Part #186002568] using a gradient solvent system with MeCNand 100 mM aqueous triethylammonium acetate. Lyopholization of theproduct fractions furnished9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purine-6-carboxamide.LCMS (ES, m/z): 701 [M−H]⁻. ¹H NMR (500 MHz, DMSO): δ 10.58 (s, 1H),9.06 (s, 1H), 8.36 (s, 1H), 8.05 (s, 1H), 7.91 (s, 1H), 7.74 (s, 1H),6.55 (br, 4H), 6.07 (d, J=7.7 Hz, 1H), 5.81 (d, J=6.0 Hz, 1H), 5.77 (m,1H), 5.04 (m, 1H), 4.96 (d, J=4.5 Hz, 1H), 4.60 (m, 1H), 4.27 (m, 1H),4.07-4.04 (m, 2H), 3.99-3.75 (m, 2H). ³¹P NMR: (202 MHz, DMSO): δ 1.9(s), −0.8 (s).

Examples 77, 78, 79, 80:2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomers 1-4)

Step 1.(2R,3R)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylhydrogen phosphonate

To a flask was added(2R,3R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite (4.00 g, 4.76 mmol), MeCN(23.65 ml), and water (0.158 ml). Pyridine trifluoroacetate (1.104 g,5.71 mmol) was added, and the reaction was stirred at rt for 1 h.Tert-butylamine (20.02 ml, 190 mmol) was then added, and stirring wascontinued at rt for 1 h, after which time the reaction was partitionedbetween hexanes and acetonitrile. The acetonitrile layer was collectedand concentrated under vacuum. DCM (39.9 ml) and water (0.798 ml) wereadded, followed by dichloroacetic acid (55.1 ml, 33.3 mmol), and thesolution was stirred for 20 min at rt, after which time triethylsilane(133 ml, 833 mmol) was added, and the reaction was stirred for a further2 h at rt. The reaction was cooled to 0° C., and pyridine (5.39 mL 66.6mmol) was added. Then the mixture was concentrated under reducedpressure to give the title compound, which was not purified further.LCMS (ES, m/z): 400 [M−H]⁻.

Step 2:O-((2R,3R,4R,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl)0-(((2S,4R,5R)-4-((hydroxyhydrophosphoryl)oxy)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl)O-hydrogenphosphorothioate

To a flask was added(2R,3R,5S)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylhydrogen phosphonate (335 mg, 0.836 mmol) and MeCN (20 mL), and then thesolution was concentrated under reduced pressure. This process wasrepeated 2×, and then MeCN (8 mL) was added, followed by activated 4 Åsieves. The mixture was stirred for 20 min at rt.(2R,3R,4R,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (825mg, 0.836 mmol) was dissolved in MeCN (5 mL). Molecular sieves (4 Å)were added, and the mixture was stirred for 30 min at rt, after whichtime this solution was transferred to the hydrogen phosphonate solutionand 2×1.5 mL portions of MeCN were used to complete the transfer. Afterstirring 30 min at rt,((dimethylamino-methylidine)amino)-3H-1,2,4-dithiazoline-3-thione (189mg, 0.919 mmol) was added. After stirring 5 min at rt, the mixture wasconcentrated under reduced pressure and purified using reverse phaseHPLC with a 10-100% gradient of MeCN and 100 mM aqueous triethylammoniumacetate. The product-containing fractions were collected andlyophilized, during which time the cyanoethyl protecting group wascleaved, to furnishO-((2R,3R,4R,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl)O-(((2S,4R,5R)-4-((hydroxyhydrophosphoryl)oxy)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl)O-hydrogenphosphorothioate. LCMS (ES, m/z): 1264 [M−H]⁻.

Step 3:N-{7-[(5S,7R,8R,12aR,14R,15R,15aR)-15-{[tert-butyl(dimethyl)silyl]oxy}-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}benzamide(Diastereomers 14)

To a flask containingO-((2R,3R,4R,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl)O-(((2S,4R,5R)-4-((hydroxyhydrophosphoryl)oxy)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl)O-hydrogenphosphorothioate (581 mg, 0.440 mmol) was added DCM (8.81 mL), water(0.079 mL, 4.40 mmol) and then dichloroacetic acid (8.74 mL, 5.28 mmol).The mixture was stirred for 15 min at rt, and then triethylsilane (10.97mL, 68.7 mmol) was added. The mixture was stirred at rt for 1.5 h andthen concentrated under reduced pressure. The mixture was dissolved inpyridine (10 mL) and then concentrated under reduced pressure. Thisprocess was repeated 2×, and then the resulting sample was dissolved inpyridine (17 ml) and 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinan-2-one(81 mg, 0.440 mmol) was added in 1 portion at rt. After stirring for 30min at rt, additional2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinan-2-one (81 mg, 0.440 mmol)was added. This sequence was repeated twice, and then water (238 μl,13.19 mmol) and 3H-1,2-benzodithiol-3-one (111 mg, 0.660 mmol) wereadded. The mixture was stirred for 1 h at rt and then partitionedbetween water (10 mL) and 1:1 EtOAc/ether (10 mL). The layers wereseparated, and the aqueous phase was extracted with 1:1 EtOAc/ether(3×10 mL). The combined organic phases were dried (MgSO₄), filtered, andconcentrated under reduced pressure. Reverse phase HPLC purification(gradient of 30-100% MeCN and 100 mM aqueous triethylammonium acetate)furnished 4 diastereomers ofN-{7-[(5S,7R,8R,12aR,14R,15R,15aR)-15-{[tert-butyl(dimethyl)silyl]oxy}-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}benzamide,all of which showed LCMS (ES, m/z): 976 [M−H]⁻.

Step 4:2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomers 1-4)

To a flask containingN-{7-[(5S,7R,8R,12aR,14R,15R,15aR)-15-{[tert-butyl(dimethyl)silyl]oxy}-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}benzamide(fastest eluting peak, 7.4 mg, 7.57 μmol) was added methylamine (33% inEtOH) (1 mL, 8.03 mmol), and the mixture was stirred at rt for 4 h,after which time the mixture was concentrated under reduced pressure.Pyridine (1 mL) was added, and the mixture was concentrated underreduced pressure. Then, pyridine (0.5 ml), triethylamine (0.104 ml,0.746 mmol) and triethylamine trihydrofluoride (0.030 ml, 0.187 mmol)were added, and the mixture was stirred at 50° C. for 16 h, after whichtime the mixture was cooled to rt and concentrated under reducedpressure. Purification by reverse phase HPLC (gradient of acetonitrileand 100 mM aqueous triethylammonium acetate) furnished Example 77,2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 1). LCMS (ES, m/z): 688 [M−H]⁻. ¹H NMR (600 MHz, DeuteriumOxide) δ 8.06 (s, 1H), 8.03 (s, 1H), 7.41 (d, J=3.8 Hz, 1H), 6.25 (d,J=3.7 Hz, 1H), 6.17 (d, J=2.6 Hz, 1H), 5.68 (d, J=7.5 Hz, 1H), 5.42-5.36(m, 2H), 5.10-5.06 (m, 1H), 4.83-4.81 (m, 1H), 4.51-4.48 (m, 1H),4.36-4.33 (m, 1H), 4.28 (dt, J=10.1, 4.9 Hz, 1H), 4.06-3.94 (m, 2H),3.03 (q, J=7.3 Hz, 12H), 2.44-2.40 (m, 2H), 1.11 (t, J=7.3 Hz, 18H).

The other diastereomers from Step 3 were individually processed in ananalogous manner to afford three additional diastereomeric products:

Example 78:2-Amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 2). LCMS (ES, m/z): 688 [M−H]⁻. ¹H NMR (600 MHz, DeuteriumOxide) δ 8.04 (s, 1H), 7.75 (s, 1H), 7.50 (d, J=3.6 Hz, 1H), 6.19-6.17(m, 1H), 6.16 (d, J=3.7 Hz, 1H), 5.65 (d, J=7.4 Hz, 1H), 5.63-5.57 (m,1H), 5.14 (td, J=7.8, 4.4 Hz, 1H), 4.54 (d, J=4.2 Hz, 1H), 4.52-4.46 (m,1H), 4.33 (d, J=8.9 Hz, 1H), 4.27 (dd, J=11.9, 3.1 Hz, 1H), 4.18-4.15(m, 1H), 3.96 (dd, J=11.4, 3.6 Hz, 2H), 3.04 (q, J=7.3 Hz, 12H),2.44-2.36 (m, 2H), 1.11 (t, J=7.3 Hz, 18H).

Example 79:2-Amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 3). LCMS (ES, m/z): 688 [M−H]⁻. ¹H NMR (600 MHz, DeuteriumOxide) δ 8.00 (s, 1H), 7.97 (s, 1H), 7.19 (d, J=3.8 Hz, 1H), 6.15 (d,J=3.8 Hz, 2H), 5.66 (d, J=7.3 Hz, 1H), 5.32 (dq, J=9.9, 7.2 Hz, 1H),4.95 (td, J=8.6, 4.6 Hz, 1H), 4.81 (d, J=4.4 Hz, 1H), 4.50 (d, J=8.0 Hz,1H), 4.32 (d, J=7.8 Hz, 1H), 4.25 (dt, J=12.0, 3.6 Hz, 1H), 4.05-3.97(m, 3H), 3.03 (q, J=7.3 Hz, 12H), 2.51-2.41 (m, 2H), 1.11 (t, J=7.3 Hz,18H).

Example 80:2-Amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 4). LCMS (ES, m/z): 688 [M−H]⁻. ¹H NMR (600 MHz, DeuteriumOxide) δ 8.00 (s, 1H), 7.72 (s, 1H), 7.23 (d, J=3.8 Hz, 1H), 6.14 (s,1H), 6.12 (d, J=3.7 Hz, 1H), 5.64 (d, J=6.9 Hz, 1H), 5.47 (dq, J=14.2,7.0 Hz, 1H), 5.05 (td, J=8.0, 4.5 Hz, 1H), 4.52 (d, J=4.4 Hz, 1H), 4.49(dt, J=7.4, 2.7 Hz, 1H), 4.33-4.27 (m, 2H), 4.19 (ddd, J=11.5, 8.5, 3.0Hz, 1H), 4.00 (dd, J=11.5, 3.9 Hz, 1H), 3.94 (ddd, J=11.6, 5.6, 2.2 Hz,1H), 3.01 (q, J=7.3 Hz, 12H), 2.49-2.40 (m, 2H), 1.10 (t, J=7.3 Hz,18H).

Example 81:2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10,16-trihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 1)

Step 1:(2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To a stirred solution of (2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite (3 g, 3.09 mmol) in MeCN (15mL) at 25° C. was added H₂O (0.111 mL, 6.18 mmol) and pyridin-1-ium2,2,2-trifluoroacetate (0.717 g, 3.71 mmol). The resulting mixture wasstirred at 25° C. for 20 min. The reaction progress was monitored byLCMS/TLC. After the phosphoramidite starting material was consumed, thereaction mixture that containing the desired product (major) was usedfor the next step without any after-treatment. LCMS (ES, m/z): 887.4[M+H]⁺.

Step 2:(2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To a stirred solution of the product of Step 1 (15.0 mL, 142 mmol) fromthe previous reaction was added tert-butylamine in one portion, and itwas stirred at 25° C. for 40 min. The resulting solution wasconcentrated in vacuo. The residue was co-evaporated with dry MeCN (twotimes, 15 mL each), used for the next step without purification. LCMS(ES, m/z): 832.3 [M−H]⁻.

Step 3:(2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To a stirred solution of the product of Step 2 in CH₂Cl₂ (37 mL) wasadded H₂O (0.558 mL, 31.0 mmol) and 6% DCA in CH₂Cl₂ (37 mL, 31.5 mmol)dropwise. The resulting mixture was stirred at 25° C. for 40 min, thenEt₃SiH (60 mL) was added, and the reaction mixture was stirred for 1.5h. Pyridine (4.5 mL, 2eq to DCA) was added to the reaction. Theresulting solution was stirred at 25° C. for 5 min and then concentratedin vacuo. The residue was triturated with MTBE/hexane (100 mL, v/v,1/1), and the supernatant was decanted. This process was repeated twomore times. The final residue was concentrated at reduced pressure andwas used for the next step without purification. LCMS (ES, m/z): 532.18[M+H]⁺.

Step 4.(2R,3R,4R,5R)-5-((((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphanyl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To a stirred solution of the product of Step 3 (0.704 g, 0.722 mmol) inMeCN (5 mL) under Ar was added activated 4 Å molecular sieve (200 mg),and the mixture was stirred at RT over 30 min.(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl (2-cyanoethyl)diisopropyl-phosphoramidite (0.822 g, 0.939 mmol) was twiceco-evaporated with dry MeCN (3 mL). Activated 4 Å molecular sieve (200mg) was added. After 30 min, the phosphoramidite solution wastransferred into the solution of the product of Step 3 by syringe. Theresulting mixture was stirred at RT for 20 min. The desired product wasdetected by TLC/LCMS, and the reaction solution was used for the nextreaction without purification. LCMS (ES, m/z): 1306.7 [M+H]⁺.

Step 5.(2R,3R,4R,5R)-5-((((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To the reaction mixture containing the product of Step 4 (˜0.722 mmol)under Ar was added(E)-N,N-dimethyl-N′-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide (163mg, 0.794 mmol) in one portion, and the mixture was stirred at RT for 30min. The reaction progress was monitored by TLC/LCMS. After theconsumption of the starting phosphite, the reaction mixture wasconcentrated in vacuo, and the residue was purified by reverse phasechromatography (X-Bridge BEH130 Prep C18) eluting with 5 to 95% MeCN inH₂O (0.04% NH₄HCO₃). The product-containing fractions were combined andconcentrated under reduced pressure to 2/3 volume. NaCl (10 g) wasadded, and the aqueous mixture was extracted with EtOAc/Et₂O (v/v, 1/1,3×80 mL). The combined organic layers were dried (Na₂SO₄) andconcentrated. LCMS (ES, m/z): 1339.5 [M+H]⁺. ³¹P-NMR (162 MHz, CD₃OD): δ67.83 (d, J=43.9 Hz), 2.81 (d, J=15.7 Hz).

Step 6.(2R,3R,4R,5R)-5-((((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To a stirred solution of the product of Step 5 (180 mg, 0.128 mmol) inCH₂Cl₂ (7 mL) was added 2,2-dichloroacetic acid in CH₂Cl₂ (2.47 mg, 1.15mmol) and H₂O (22.97 mg, 1.28 mmol). After stirring at RT for 20 min,Et₃SiH (4.5 mL) was added. After 2 h, pyridine (1 mL) was added, and themixture was stirred for 10 min. After removal of volatiles, the productwas used for the next reaction step without purification. LCMS (ES,m/z): 1036.4 [M+H]⁺.

Step 7:(5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-15-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate2-sulfide

The product of Step 6 (570 mg) was co-evaporated with dry pyridine (1 mLeach, three times). To the mixture in dry pyridine (4 mL) at RT under Arwas added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (71 mg,0.384 mmol) in one portion. The resulting mixture was stirred for 40min. The reaction progress was monitored by TLC/LCMS. The desiredproduct as a mixture of diastereomers was observed, and the product wasused for the next reaction step directly. LCMS (ES, m/z): 1018.5 [M+H]⁺.

Step 8: Diastereomeric mixtures(5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-15-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-10-thiolate10-oxide 2-sulfide (A1) and(5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-15-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-10-thiolate10-oxide 2-sulfide (A2)

To the stirred mixture of the product of Step 7 was added H₂O (69.2 mg,3.84 mmol) and 3H-benzo[c][1,2]dithiol-3-one (32.3 mg, 0.192 mmol). Themixture was stirred at RT for 40 min. The reaction progress wasmonitored by TLC/LCMS. After the reaction completed, the reactionmixture was poured into aq NaHCO₃ (0.14 g NaHCO₃ in 5 mL H₂O) andstirred for 5 min. The resulting mixture was extracted with EtOAc/ether(v/v, 1/1, 3×15 mL). The combined organic layers were dried (Na₂SO₄),and purified by silica gel chromatography eluted with 0 to 15% MeOH inCH₂Cl₂ to give products: mixture of diastereomers A1 (eluted out at 5.5%MeOH in CH₂Cl₂); mixture of diastereomers A2 (eluted out at 9.8% MeOH inCH₂Cl₂);(2R,3R,4R,5R)-5-((((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate (eluted out at 12.6% MeOH in CH₂Cl₂). Mixture A1: LCMS (ES,m/z): 1050.30 [M+H]⁺. ³¹P-NMR (162 MHz, CD₃OD): δ 66.34 (s), 64.63 (s).Mixture A2: LCMS (ES, m/z): 1050.30 [M+H]⁺. ³¹P-NMR (162 MHz, CD₃OD): δ65.94, 64.17, 62.55, 61.28.

Step 9: Diastereomers(5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate)2,10-dioxide (BI) and(5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate)2,10-dioxide (B2)

To a stirred suspension of crude A1 (95 mg, ˜0.09 mmol) from theprevious step in MeCN (1 mL) at RT under Ar was added tert-butylamine(1.5 mL). After 30 min, volatile components were removed in vacuo. Theresidue was purified by reverse phase prep-HPLC (X-Bridge BEH130 PrepC18) eluted with 25 to 45% MeCN in aq NH₄HCO₃ (10 mM) over 8 min to givecompound B2 as a single diastereomer (T_(R)=5.97 min). LCMS (ES, m/z):891.4 [M−H]⁻. H-NMR (300 MHz, CD₃OD): δ 8.44 (s, 1H), 8.22 (s, 1H), 8.09(s, 1H), 6.37 (d, J=14.0 Hz, 1H), 5.95 (d, J=8.0 Hz, 1H), 5.65-5.54 (m,1H), 5.26-5.10 (m, 2H), 4.57-4.41 (m, 4H), 4.24 (s, 1H), 4.01 (d, J=11.5Hz, 1H), 3.89 (d, J=11.8 Hz, 1H), 2.75-2.63 (m, 1H), 1.04-0.91 (m, 15H),0.28-0.24 (m, 6H). ³¹P-NMR (121 MHz, CD₃OD): δ 57.10 (s), 53.1 (s).

Step 10:(5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-6-{[tert-butyl(dimethyl)silyl]oxy}-15-fluorooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,O-bis(thiolate) 2,10-dioxide

To a stirred solution of compound B2 (13 mg, 0.013 mmol) from theprevious step was added a solution of MeNH₂ in EtOH (0.6 mL, 30% byweight). The mixture was stirred at RT for 12 h. The volatile componentswere removed under reduced pressure, and the residue containing productcompound was used for the next reaction step without purification. LCMS(ES, m/z): 823.15 [M+H]⁺.

Step 11:2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10,16-trihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 1)

The crude product from Step 10 (25 mg) was co-evaporated withpyridine/Et₃N (v/v, 3/1, 1 mL each, three times) and then dissolved inpyridine (0.15 mL). The mixture was charged with Ar and Et₃N (0.20 mL)and triethylamine trihydrofluoride (56.4 mg, 0.350 mmol) were added. Theresulting solution was warmed at 50° C. for 6 h. The reaction progresswas monitored by TLC/LCMS. After completion of the reaction, the mixturewas concentrated in vacuo and then, co-evaporated with MeCN (threetimes, 1 mL each). The residue was purified by reverse phase prep-HPLC(X-Bridge BEH130 Prep C18) eluted with 0 to 22% MeCN in aq NH₄HCO₃ (50mM) over 15 min to give the product compound (T_(R)=8.3 min). LCMS (ES,m/z): 708.95 [M+H]⁺. ¹H-NMR (400 MHz, D₂O): δ 8.18 (s, 1H), 8.16 (s,1H), 7.77 (s, 1H), 6.37 (d, J=14.3 Hz, 1H), 5.86 (d, J=8.4 Hz, 1H),5.61-5.54 (m, 1.5H), 5.43 (s, 0.5H), 5.27-5.12 (m, 2H), 4.59 (d, J=3.6Hz, 1H), 4.47 (t, J=12.9 Hz, 2H), 4.36 (d, J=4.8 Hz, 1H), 4.04 (dd,J=23.2, 12.0 Hz, 2H). ³¹P-NMR (162 MHz, D₂O): δ 55.63 (s), 51.55 (s).

Example 82:2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10,16-trihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 2)

Step 1: Diastereomers(5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate)2,10-dioxide (B3) and(5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate)2,10-dioxide (B4)

To a stirred suspension of crude A2 (105 mg, ˜0.1 mmol) from Example 81,Step 8 in MeCN (1 mL) under Ar was added tert-butylamine (1.5 mL), andthe mixture was stirred at RT for 30 min. The volatile components wereremoved in vacuo. The residue was purified by reverse phase prep-HPLC(X-Bridge BEH130 Prep C18) eluted with 25 to 40% MeCN in aq NH₄HCO₃ (10mM) over 10 min to give two diastereomeric compounds, B3 (T_(R)=6.12min, 0.025 mmol) and B4 (T_(R)=7.45 min, 0.021 mmol).

Compound B3: LCMS (ES, m/z): 995.3 [M−H]⁻. H-NMR (300 MHz, CD₃OD): δ8.82 (s, 1H), 8.72 (s, 1H), 8.65 (s, 1H), 8.20-8.13 (m, 2H), 7.66-7.54(m, 3H), 6.47 (d, J=14.0 Hz, 1H), 6.09 (d, J=8.4 Hz, 1H), 5.96-5.95 (m,0.5H), 5.81-5.78 (m, 0.5H), 5.52-5.36 (m, 2H), 4.64-4.56 (m, 2H),4.48-4.43 (m, 1H), 4.37-4.30 (m, 1H), 4.25-4.22 (m, 1H), 4.17-4.10 (m,1H), 3.98 (d, J=11.7 Hz, 1H), 2.65 (p, J=6.8 Hz, 1H), 1.12 (d, J=6.8 Hz,3H), 0.98-0.95 (m, 12H), 0.22 (d, J=8.0 Hz, 6H). ³¹P-NMR (121 MHz,CD₃OD): δ 56.96 (s), 55.90 (s).

Compound B4: LCMS (ES, m/z): 995.4 [M−H]⁻. H-NMR (300 MHz, CD₃OD): δ8.97 (s, 1H), 8.68 (s, 1H), 8.24-8.22 (m, 3H), 7.59 (ddd, J=14.5, 7.9,6.2 Hz, 3H), 6.46 (d, J=13.0 Hz, 1H), 5.99 (d, J=8.3 Hz, 1H), 5.67-5.57(m, 1H), 5.45-5.33 (m, 2H), 4.56 (dd, J=13.5, 4.9 Hz, 2H), 4.47-4.38 (m,2H), 4.25 (t, J=3.5 Hz, 1H), 4.07 (d, J=11.3 Hz, 1H), 3.94 (d, J=11.0Hz, 1H), 2.75 (p, J=6.8 Hz, 1H), 1.04 (d, J=6.8 Hz, 3H), 0.95 (s, 12H),0.23 (d, J=5.2 Hz, 6H). ³¹P-NMR (121 MHz, CD₃OD): δ 56.81 (s), 54.76(s).

Step 2:(5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluorooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,O-bis(thiolate) 2,10-dioxide

Compound B3 from Step 1 (25 mg, 0.025 mmol) was dissolved in a solutionof MeNH₂ in EtOH (1 mL, 30% by weight), and the mixture was stirred atRT for 12 h. The reaction progress was monitored by TLC/LCMS. After thereaction was complete, the volatile components were removed in vacuo,and the residue containing the crude product was used for the nextreaction step without purification. LCMS (ES, m/z): 823.25 [M+H]⁺.

Step 3:2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10,16-trihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 2)

The crude product of Step 2 (35 mg) was co-evaporated with pyridine/Et₃N(v/v, 3/1, 1 mL each, three times) and then re-dissolved in pyridine(0.4 mL). The mixture was charged with Ar and Et₃N (0.34 mL, 2.4 mmol),and triethylamine trihydrofluoride (97 mg, 0.6 mmol) were added.

The resulting solution was warmed at 50° C. for 6 h. Then, the mixturewas concentrated at reduced pressure and then co-evaporated with MeCN(3×1 mL). The residue was purified by reverse phase prep-HPLC (X-BridgeBEH130 Prep C18) eluted with 0 to 10% MeCN in aq NH₄HCO₃ (50 mM) over 14min to give the product compound (T_(R)=9.2 min). LCMS (ES, m/z): 709.00[M+H]⁺. H-NMR (400 MHz, DMF-d₇+D₂O): δ 8.68 (s, 1H), 8.61 (s, 1H), 8.45(s, 1H), 6.57 (d, J=14.8 Hz, 1H), 6.27-6.25 (m, 1.5H), 6.15-6.13 (m,0.5H), 5.72-5.68 (m, 1H), 5.56-5.54 (m, 1H), 4.85-4.83 (m, 1H),4.71-4.69 (m, 1H), 4.52-4.43 (m, 4H), 4.27-4.24 (m, 1H). ³¹P-NMR (162MHz, DMF-d₇+D₂O): δ 56.03 (s), 53.37 (s). ¹⁹F-NMR (376 MHz, DMF-d₇+D₂O):δ −205.44 (s).

Example 83:2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10,16-trihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 3)

Step 1:(5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluorooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate)2,10-dioxide

Compound B4((5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluorooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate)2,10-dioxide, 21 mg, 0.021 mmol) from Example 82, Step 1 was dissolvedin a solution of MeNH₂ in EtOH (1 mL, 30% by weight), and the mixturewas stirred at RT for 12 h. The reaction progress was monitored byTLC/LCMS. After the reaction was complete, the volatile components wereremoved in vacuo, and the product was used for the next reaction stepwithout purification. LCMS (ES, m/z): 823.25 [M+H]⁺.

Step 2:2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10,16-trihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 3)

The crude product of Step 1 (31 mg) was co-evaporated with pyridine/Et₃N(v/v, 3/1, 3×1 mL) and then, re-dissolved in pyridine (0.4 mL). Themixture was charged with Ar and Et₃N (0.28 mL, 2.0 mmol) andtriethylamine trihydrofluoride (81 mg, 0.5 mmol) were added. Theresulting solution was warmed at 50° C. for 6 h. The mixture wasconcentrated at reduced pressure and then co-evaporated with MeCN (3×1mL). The residue was purified by reverse phase prep-HPLC (X-BridgeBEH130 Prep C18) eluting with 0 to 10% MeCN in aq NH₄HCO₃ (50 mM) over14 min to give the product compound (T_(R)=10.1 min). LCMS (ES, m/z):709.00 [M+H]⁺. H-NMR (400 MHz, DMF-d₇+D₂O): δ 8.73 (s, 1H), 8.28-8.20(m, 2H), 6.55 (d, J=14.8 Hz, 1H), 6.25-5.85 (m, 3H), 5.62-5.56 (m, 1H),4.76 (s, 1H), 4.62-4.60 (m, 2H), 4.49-4.41 (m, 3H), 4.18-4.15 (m, 1H).³¹P-NMR (162 MHz, DMF-d₇+D₂O): δ 56.09 (s), 54.75 (s). ¹⁹F-NMR (376 MHz,DMF-d₇+D₂O): δ −203.33 (s).

Examples 84 through 116, shown in Table 5 below, were prepared accordingto procedures analogous to those outlined in Examples 77 through 83above using the appropriate monomers, described as Preparations or asobtained from commercial sources, in the coupling step.

TABLE 5 Mass Ex. Structure Name [M − H]⁻  84

2-amino-9-[(5R,7R,8R,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-2,10,16-trihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 1) 691 [M + H]⁺  85

2-amino-9-[(5R,7R,8R,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-2,10,16-trihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 2) 691 [M + H]⁺  86

2-amino-9-[(2R,5S,7R,8R,10R, 12aR,14R,15R,15aS)-14-(6-amino-9H-purin-9-yl)-2,10,15-trihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 1) 689  87

2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(6-amino-9H-purin-9-yl)-2,10,15-trihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 2) 689  88

2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(6-amino-9H-purin-9-yl)-2,10,15-trihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 3) 689  89

2-amino-9-[(2R,5R,7R,8R,10,R, 12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10,16-trihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 1) 707  90

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10,16-trihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 2) 707  91

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10,16-trihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 3) 707  92

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10,16-trihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 4) 707  93

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6-amino-9H-purin-9-yl)-2,10,18-trihydroxy-2,10-disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 717  94

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6-amino-9H-purin-9-yl)-2,10,18-trihydroxy-2,10-disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 717  95

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6-amino-9H-purin-9-yl)-2,10,18-trihydroxy-2,10-disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3) 719 [M + H]⁺  96

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6-amino-9H-purin-9-yl)-15-fluoro-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 1) 723  97

2-amino-9-[(2R,5R,7R,8R,10R, 12aR,14R,15S,15aR,16S)-14-(6-amino-9H-purin-9-yl)-15-fluoro-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 2) 723  98

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6-amino-9H-purin-9-yl)-15-fluoro-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 3) 723  99

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6-amino-9H-purin-9-yl)-15-fluoro-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 4) 723 100

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-2,10,18-trihydroxy-2,10-disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7(12H)-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 1)733 101

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-2,10,18-trihydroxy-2,10-disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclo-tetradecin-7(12H)-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 2) 733102

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-2,10,18-trihydroxy-2,10-disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3) 733 103

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aS,16S)-14-(6-amino-9H-purin-9-yl)-12a-ethynyl-16-fluoro-2,10,15-trihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 1) 731 104

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aS,16S)-14-(6-amino-9H-purin-9-yl)-12a-ethynyl-16-fluoro-2,10,15-trihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 2) 731 105

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aS,16S)-14-(6-amino-9H-purin-9-yl)-12a-ethynyl-16-fluoro-2,10,15-trihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 3) 731 106

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-12a-ethynyl-16-fluoro-2,10,15-trihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 1) 731 107

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-12a-ethynyl-16-fluoro-2,10,15-trihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 2) 731 108

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7- one (Diastereomer 1) 706109

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7- one (Diastereomer 2) 706110

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7- one (Diastereomer 3) 706111

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7- one (Diastereomer 1) 706112

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7- one (Diastereomer 2) 706113

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7- one (Diastereomer 3) 706114

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7- one (Diastereomer 4) 706115

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15-fluoro-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-3,6-dihydro- 7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 1) 725 116

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15-fluoro-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-3,6-dihydro- 7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 2) 725

Examples 117 and 118:2-amino-9-[(2R,5R,7R,8S,10R,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 1) and2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 2)

Step 1:2R,3S,4R,5R)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphanyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite(1058 mg, 1.234 mmol) was co-evaporated with dry ACN (3×5 mL),re-dissolved in ACN (10 mL) under Ar, and dried by adding activated 4 Åmolecular sieve (200 mg).(2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate (430 mg, 1.03 mmol) and pyridinium 2,2,2-trifluoroacetate(298 mg, 1.54 mmol) were co-evaporated with ACN (3×5 mL) and thenre-dissolved in ACN (10 mL), and dried by adding activated 4 Å molecularsieve (200 mg). After 30 min, it was added to the previously preparedmixture containing((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl (2-cyanoethyl)diisopropylphosphoramidite. It was stirred at rt for 30 min. Thereaction mixture was used for the next reaction step withoutpurification. LCMS (ES, m/z): 1173.8 [M−H]⁻.

Step 2.(2R,3S,4R,5R)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To the reaction mixture from Step 1 was added(E)-N,N-dimethyl-N-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide(DDTT, 0.232 g, 1.13 mmol) in one portion. The mixture was stirred at rtfor 1 h. It was concentrated to give a crude sample containing theproduct, which was used for the next reaction step without purification.LCMS (ES, m/z): 1205.8 [M−H]⁻.

Step 3.(2R,3S,4R,5R)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To a solution of the crude from Step 2 in CH₂Cl₂ (15 mL) was added water(0.2 mL, 10 mmol) and 2,2-dichloroacetic acid in CH₂Cl₂ (0.6M, 15 mL, 9mmol). After 30 min, triethylsilane (28 mL) was added, and it wasstirred for 1.5 h. Then, pyridine (1.4 mL) was added. It wasconcentrated, and the residue was purified by reverse phase (C18)chromatography eluted with 0 to 43% ACN in aq NH₄HCO₃ (5 mM) to give theproduct. LCMS (ES, m/z): 905.8 [M+H]⁺. ¹H-NMR (300 MHz, CD₃OD): δ8.71-8.44 (m, 2H), 8.21-8.03 (m, 3H), 7.80 (d, J=10.4 Hz, 0.5H),7.66-7.61 (m, 1H), 7.54 (t, J=7.6 Hz, 2H), 6.61-6.42 (m, 1H), 6.14 (dd,J=13.2, 6.0 Hz, 1H), 5.68 (d, J=9.9 Hz, 0.5H), 5.60-5.19 (m, 3H),4.69-4.36 (m, 3H), 4.36-4.17 (m, 3H), 3.92-3.64 (m, 2H), 3.13-2.55 (m,5H), 1.19 (dd, J=6.9, 2.1 Hz, 6H). ¹⁹F-NMR (282 MHz, CD₃OD): δ −202.55,−202.75 (d, 1F). ³¹P-NMR (121 MHz, CD₃OD): δ 66.91, 66.69 (2s, 1P);2.66, 2.60 (2s, 1P).

Step 4:(5R,7R,8S,12aR,14R,15aS,16R)-2-(2-cyanoethoxy)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate2-sulfide

Diphenyl phosphorochloridate (2375 mg, 8.84 mmol) was added to pyridine(45 ml) at −30° C. To the solution at −30° C. was added(2R,3S,4R,5R)-5-((((((2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate (400 mg, 0.44 mmol) in CH₂Cl₂ (45 mL) dropwise over 20 min.The resulting mixture was stirred at −30° C. for 40 min. The reactionmixture was used for the next reaction step immediately withoutpurification. LCMS (ES, m/z): 887.8 [M+H]⁺.

Step 5:(5R,7R,8S,12aR,14R,15aS,16R)-2-(2-cyanoethoxy)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate2,10-disulfide

To the mixture from Step 4 at −30° C. was added3H-benzo[c][1,2]dithiol-3-one (112 mg, 0.663 mmol) and water (279 μL,15.5 mmol). After stirring at rt for 1 h, the mixture was poured into asolution of Na₂S₂O₃.5H₂O (280 mg) in water (10 mL) at 0° C. It wasstirred at rt for 5 min, and the mixture was concentrated under reducedpressure. The residue was purified by reverse phase (C18) chromatographyeluted with 0 to 28% ACN in aq NH₄HCO₃ (5 mM) to give the product. LCMS(ES, m/z): 919.8 [M+H]⁺. ¹⁹F-NMR (376 MHz, CD₃OD): δ −198.51, −198.98,−200.16 (3s, 1F)⁺. ³¹P-NMR (162 MHz, CD₃OD): δ 65.90, 65.09, 63.64,62.95, 57.26, 56.50 (6s, 2P).

Step 6. Diastereomers(5R,7R,8S,12aR,14R,15aS,16R)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate2,10-disulfide (28-6-A),(5R,7R,8S,12aR,14R,15aS,16R)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate2,10-disulfide (28-6-B), and(5R,7R,8S,12aR,14R,15aS,16R)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate2,10-disulfide (28-6-C)

To a solution of (5R,7R,8S,12aR,14R,15aS,16R)-2-(2-cyanoethoxy)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate2,10-disulfide (265 mg, 0.288 mmol) in ACN (5 mL) at rt was addedtert-butylamine (5 mL, 0.29 mmol). The reaction mixture was stirred for10 min. Then, volatile components were removed under reduced pressure.The residue was purified by preparative-HPLC (T3 Prep Column, 19 mm×250mm) eluted with 5 to 20% ACN in aq NH₄HCO₃ (50 mM) over 21 min.

The first fractions (T_(R): 8.95 min) gave(5R,7R,8S,12aR,14R,15aS,16R)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate2,10-disulfide (28-6-A). LCMS (ES, m/z): 866.7 [M+H]⁺. ¹H-NMR (400 MHz,DMSO-d₆): δ 8.74 (s, 1H), 8.71 (s, 1H), 8.26 (s, 1H), 8.07 (d, J=7.5 Hz,1H), 7.65 (d, J=7.7 Hz, 1H), 7.57 (t, J=7.5 Hz, 2H), 6.60-6.34 (m, 1H),5.94 (d, J=8.6 Hz, 1H), 5.91-5.66 (m, 1H), 5.46-5.16 (m, 2H), 4.50 (d,J=27.0 Hz, 1H), 4.27 (d, J=9.8 Hz, 1H), 4.16 (t, J=10.1 Hz, 1H), 3.98(q, J=11.0 Hz, 1H), 3.86 (d, J=11.9 Hz, 1H), 3.72-3.69 (m, 1H),3.10-3.06 (m, 1H), 3.00-2.82 (m, 1H), 2.74-2.70 (m, 1H), 1.06 (dd,J=27.2, 6.8 Hz, 6H). ³¹P-NMR (162 MHz, DMSO-d₆): δ 53.92 (s, 1P), 52.99(s, 1P).

The second fractions (T_(R): 10.00 min) gave(5R,7R,8S,12aR,14R,15aS,16R)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate2,10-disulfide (28-6-B). LCMS (ES, m/z): 866.7 [M+H]⁺.

The third fractions (T_(R): 11.27-12.16 min) gave(5R,7R,8S,12aR,14R,15aS,16R)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate2,10-disulfide (28-6-C), a mixture of two diastereomers, which was usedin next step. LCMS (ES, m/z): 866.7 [M+H]⁺. ¹H-NMR (400 MHz, DMSO-d₆): δ8.77 (s, 1H), 8.74 (s, 1H), 8.72 (s, 1H), 8.12-8.02 (m, 2H), 7.66-7.64(m, 1H), 7.56 (t, J=7.5 Hz, 2H), 6.47-6.44 (m, 1H), 5.99 (d, J=8.6 Hz,1H), 5.55-5.33 (m, 2H), 5.22 (d, J=11.6 Hz, 1H), 4.47 (d, J=25.7 Hz,1H), 4.43-4.40 (m, 1H), 4.03-3.98 (m, 2H), 3.84 (d, J=11.8 Hz, 1H),3.75-3.72 (m, 1H), 3.18-3.15 (m, 1H), 2.82-2.73 (m, 2H), 1.13 (dd,J=6.9, 2.5 Hz, 6H). ³¹P-NMR (162 MHz, DMSO): δ 53.42 (s, 1P), 52.16 (s,1P).

Step 7:2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomers 1 and 2)

(5R,7R,8S,12aR,14R,15aS,16R)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate2,10-disulfide (28-6-C) (180 mg, 0.208 mmol) was dissolved in a solutionof MeNH₂ in EtOH (30%, 5.0 mL, 42 mmol), and the resulting solution wasstirred at rt for 1 h. The volatile components were removed underreduced pressure to give a crude sample that was purified by Prep-HPLC(Atlantis Prep T3 OBD Column, 19 mm×250 mm) eluted with 5 to 19.5% ACNin aq NH₄HCO₃ (50 mM) over 19 min to give, after concentration:

Example 117 (T_(R): 14.82 min):2-amino-9-[(2R,5R,7R,8S,10R,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 1). LCMS (ES, m/z): 690.8 [M−H]⁻. ¹H-NMR (400 MHz, D₂O): δ8.14 (s, 1H), 8.13 (s, 1H), 8.11 (s, 1H), 6.37 (t, J=5.5 Hz, 1H), 5.99(d, J=8.7 Hz, 1H), 5.54 (d, J=3.3 Hz, 0.5H), 5.48-5.30 (m, 1.5H), 5.12(dd, J=10.2, 5.5 Hz, 1H), 4.66 (d, J=34.1 Hz, 1H), 4.36 (s, 1H),4.24-4.01 (m, 4H), 3.04 (dt, J=14.1, 5.6 Hz, 1H), 2.79 (dt, J=13.5, 6.4Hz, 1H). ¹⁹F-NMR (376 MHz, D₂O): δ −198.66 (s, 1F). ³¹P-NMR (162 MHz,D₂O): δ 53.97 (s, 1P), 53.46 (s, 1P).

Example 118 (T_(R): 15.93 min): 2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 2). LCMS (ES, m/z): 690.8 [M−H]⁻. ¹H-NMR (400 MHz, D₂O): δ8.16 (s, 1H), 8.11 (s, 1H), 7.83 (s, 1H), 6.34 (dd, J=6.5, 3.0 Hz, 1H),5.95 (d, J=8.6 Hz, 1H), 5.69-5.53 (m, 1H), 5.47 (d, J=3.4 Hz, 0.5H),5.33 (d, J=3.4 Hz, 0.5H), 5.23 (p, J=7.3 Hz, 1H), 4.64 (d, J=26.7 Hz,1H), 4.35 (ddd, J=10.6, 6.9, 3.2 Hz, 1H), 4.31-4.17 (m, 2H), 4.05-3.95(m, 2H), 2.94-2.85 (m, 1H), 2.74 (dt, J=14.0, 7.2 Hz, 1H). ¹⁹F-NMR (376MHz, D₂O): δ −198.74 (s, 1F). ³¹P-NMR (162 MHz, D₂O): δ 55.05 (s, 1P),52.87 (s, 1P).

Example 119:2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12_(H)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 3)

Step 1:2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

(5R,7R,8S,12aR,14R,15aS,16R)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate2,10-disulfide (28-6-B) (45 mg, 0.053 mmol) was dissolved in a solutionof MeNH₂ in EtOH (30%, 1.5 mL, 11 mmol), and the resulting solution wasstirred at rt for 1 h. The volatile components were removed underreduced pressure, and the residue was purified by Prep-HPLC (AtlantisPrep T3 OBD Column, 19 mm×250 mm) eluted with 18 to 19.5% ACN in aqNH₄HCO₃ (50 mM) over 16 min to give the product (T_(R): 11.22 min). LCMS(ES, m/z): 690.8 [M−H]⁻. ¹H-NMR (400 MHz, D₂O): δ 8.32 (s, 1H), 8.15 (s,1H), 7.96 (s, 1H), 6.41 (t, J=5.7 Hz, 1H), 6.00 (d, J=8.6 Hz, 1H), 5.56(dt, J=22.9, 10.4 Hz, 1H), 5.40-5.30 (m, 1.5H), 5.19 (d, J=3.6 Hz,0.5H), 4.64 (d, J=28.3 Hz, 1H), 4.40-4.27 (m, 2H), 4.27-4.17 (m, 1H),4.02 (d, J=11.9 Hz, 1H), 3.95-3.85 (m, 1H), 2.92 (dt, J=14.1, 5.6 Hz,1H), 2.79 (td, J=13.8, 13.1, 6.1 Hz, 1H). ¹⁹F-NMR (376 MHz, D₂O): δ−198.02 (s, 1F). ³¹P-NMR (162 MHz, D₂O): δ 57.89 (s, 1P), 55.05 (s, 1P).

Example 120:2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12_(H)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 4)

Step 1:2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

(5R,7R,8S,12aR,14R,15aS,16R)-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-diolate2,10-disulfide (28-6-A) (45 mg, 0.053 mmol) was dissolved in a solutionof MeNH₂ in EtOH (30%, 1.5 mL, 11 mmol), and the resulting solution wasstirred at rt for 1 h. The volatile components were removed underreduced pressure, and the residue was purified by Prep-HPLC (AtlantisPrep T3 OBD Column, 19 mm×250 mm) eluted with 4 to 11% ACN in aq NH₄HCO₃(50 mM) over 17 min to give the product (T_(R): 11.72 min). LCMS (ES,m/z): 690.8 [M−H]⁻. ¹H-NMR (400 MHz, D₂O): δ 8.33 (s, 1H), 8.29 (s, 1H),8.16 (s, 1H), 6.41 (t, J=6.5 Hz, 1H), 6.05 (d, J=8.6 Hz, 1H), 5.49-5.32(m, 1.5H), 5.23 (d, J=3.5 Hz, 0.5H), 4.79-4.73 (m, 1H), 4.69-4.59 (m,1H), 4.40-4.32 (m, 1H), 4.23 (q, J=8.9, 7.5 Hz, 2H), 4.07 (d, J=11.8 Hz,1H), 3.94-3.84 (m, 1H), 3.00 (dt, J=12.7, 6.2 Hz, 1H), 2.94-2.84 (m,1H). ¹⁹F-NMR (376 MHz, D₂O): δ −197.92 (s, 1F). ³¹P-NMR (162 MHz, D₂O):δ 59.46 (s, 1P), 54.42 (s, 1P).

Example 121, 122, 123:2-amino-9-[(2R,5S,7R,8R,10R,12aR,14R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 1) and2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomers 2 and 3)

Step 1.(2R,3R,5S)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylhydrogen phosphonate

To a flask was added (2R,3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite (4 g, 4.76 mmol), MeCN (23.65ml), and water (0.158 ml). Pyridine trifluoroacetate (1.104 g, 5.71mmol) was then added, and the mixture was stirred 1 h at rt, after whichtime tert-butylamine (20.02 ml, 190 mmol) was added. After stirring 1 hat rt, the mixture was concentrated under reduced pressure. Theresulting mixture was dissolved in DCM (39.9 ml), and then water (0.798ml) was added, followed by dichloroacetic acid (2.75 ml, 33.3 mmol). Thesolution was stirred for 20 min at rt, and then triethylsilane (133 ml,833 mmol) was added, and the reaction was stirred for a further 2 h atrt. After cooling to 0° C., pyridine was added, and the mixture wasconcentrated under reduced pressure. The resulting sample waspartitioned between hexanes (100 mL) and water (20 mL). The layers wereseparated, and the aqueous phase was purified by reverse phase HPLCusing a gradient solvent system of 0-35% MeCN in 0.04% aq ammoniumbicarbonate. The product-containing fractions were collected andlyophilized to give(2R,3R,5S)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylhydrogen phosphonate. LCMS (ES, m/z): 400 [M−H]⁻.

Step 2:(2R,3S,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl (2-cyanoethyl)diisopropylphosphoramidite

To a flask was addedN-(7-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide(4 g, 6.09 mmol), DCM (71.7 ml), and 4,5-dicyanoimidazole (2.158 g,18.27 mmol), and the solution was cooled to 0° C. 2-CyanoethylN,N,N′,N′-tetraisopropylphosphorodiamidite (6.77 ml, 21.32 mmol) wasadded, and the mixture was stirred for 15 min at 0° C., after which timethe mixture was concentrated under reduced pressure and purified bysilica gel chromatography using a gradient of 30-100% EtOAc in hexanesto yield(2R,3S,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite. LCMS (ES, m/z): 857 [M+H]⁻.

Step 3:(2R,3R,5S)-5-((((((2R,3S,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylhydrogen phosphonate

To a flask containing(2R,3R,5S)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylhydrogen phosphonate (1.62 g, 2.81 mmol) was added pyridinetrifluoroacetate (0.543 g, 2.81 mmol), activated 4 Å sieves and MeCN (10mL), and the mixture was stirred 20 min at rt. To a separate flaskcontaining(2R,3S,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (2.408 g,2.81 mmol) was added MeCN (10 mL) and activated 4 Å sieves, and themixture was stirred 20 min at rt, after which time the hydrogenphosphonate solution was added, and MeCN (2×4 mL) was used to completethe transfer. The mixture was stirred 1 h at rt, and then((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazoline-3-thione (0.634g, 3.09 mmol) was added. The resulting mixture was stirred 30 min at rtand then concentrated under reduced pressure. Reverse phase HPLCpurification using a gradient solvent system of 5-100% MeCN in 0.04%aqueous ammonium bicarbonate yielded(2R,3R,5S)-5-((((((2R,3S,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylhydrogen phosphonate. LCMS (ES, m/z): 1187 [M−H]⁻.

Step 4:N-{7-[(5S,7R,8R,12aR,14R,15aS)-2-(2-cyanoethoxy)-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-10-oxido-10-sulfanyl-2-sulfidooctahydro-12H-5,8-methanofuro3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}benzamide

To a flask containing (2R,3R,5 S)-5-((((((2R,3S,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-2-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-ylhydrogen phosphonate (1.34 g, 1.127 mmol) was added DCM (22.54 ml) andwater (0.203 ml, 11.27 mmol), and then dichloroacetic acid (1.116 ml,13.52 mmol) was added. The solution was stirred for 20 min at rt, andthen triethylsilane (28.1 ml, 176 mmol) was added. After stirring 3 h atrt, the mixture was concentrated under reduced pressure. Pyridine (50mL) was added, and then the mixture was concentrated under reducedpressure. This process was repeated 2×, and then pyridine (37.6 ml) wasadded, followed by 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane2-oxide (0.624 g, 3.38 mmol). The resulting mixture was stirred for 1 hat rt, after which time water (610l, 33.8 mmol) was added, followed by3H-1,2-benzodithiol-3-one (285 mg, 1.692 mmol). The resulting mixturewas stirred 30 min at rt and then concentrated under reduced pressure.HPLC purification using a gradient solvent system of MeCN in 100 mMaqueous triethylammonium acetate yielded 3 separate diastereomers ofN-{7-[(5S,7R,8R,12aR,14R,15aS)-2-(2-cyanoethoxy)-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl})-10-oxido-10-sulfanyl-2-sulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}benzamideafter lyopholization, all of which showed LCMS (ES, m/z): 899 [M−H]*.

Step 5:2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

To a vial containing one diastereomer (slowest eluting) ofN-{7-[(5S,7R,8R,12aR,14R,15aS)-2-(2-cyanoethoxy)-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-10-oxido-10-sulfanyl-2-sulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}benzamide(122 mg, 0.135 mmol) was added methylamine (33% in EtOH) (2 mL, 16.07mmol) and the resulting solution was stirred for 4 h at rt, after whichtime it was concentrated under reduced pressure. Purification by reversephase HPLC using a gradient solvent system of MeCN in 100 mM aqueoustriethylammonium acetate yielded Example 121,2-amino-9-[(2R,5S,7R,8R,10R,12aR,14R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 1). LCMS (ES, m/z): 672 [M−H]⁻. ¹H NMR (600 MHz, DMSO-d₆)δ 10.54 (s, 1H), 8.01 (s, 1H), 7.86 (s, 1H), 7.29 (d, J=2.7 Hz, 1H),6.97 (s, 2H), 6.60-6.53 (m, 3H), 6.44 (t, J=6.9 Hz, 1H), 5.73 (d, J=2.7Hz, 1H), 4.95 (s, 1H), 4.80 (s, 1H), 4.38-4.27 (m, 2H), 3.97-3.91 (m,2H), 3.90-3.82 (m, 1H), 3.64 (dt, J=16.1, 9.3 Hz, 1H), 2.69-2.63 (m,1H), 2.63 (s, 12H), 2.60-2.54 (m, 3H), 2.22-2.13 (m, 1H), 1.01-0.94 (m,18H).

The other diastereomers from Step 4 were treated in an analogous mannerto produce two additional diastereomers of2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one:

Example 122 (Diastereomer 2): LCMS (ES, m/z): 672 [M−H]⁻. ¹H NMR (600MHz, DMSO-d₆) δ 10.57 (s, 1H), 9.55 (s, 2H), 8.10 (s, 1H), 7.95 (s, 1H),7.47 (s, 1H), 7.42 (d, J=3.2 Hz, 1H), 6.66 (d, J=3.4 Hz, 1H), 6.51 (s,2H), 6.49-6.44 (m, 1H), 5.73 (d, J=4.0 Hz, 1H), 5.08-5.00 (m, 2H),4.31-4.27 (m, 1H), 4.22 (t, J=11.3 Hz, 1H), 4.07 (q, J=10.6 Hz, 1H),4.00-3.95 (m, 1H), 3.76-3.71 (m, 1H), 3.65 (td, J=11.6, 6.1 Hz, 1H),3.02 (d, J=4.2 Hz, 12H), 2.67-2.61 (m, 1H), 2.61-2.53 (m, 2H), 2.18 (dt,J=11.7, 5.3 Hz, 1H), 1.12 (t, J=7.3 Hz, 18H).

Example 123 (Diastereomer 3): LCMS (ES, m/z): 672 [M−H]⁻. ¹H NMR (600MHz, DMSO-d₆) δ 10.53 (s, 1H), 8.01 (s, 3H), 7.28 (d, J=3.4 Hz, 1H),6.97 (s, 2H), 6.56 (d, J=3.5 Hz, 1H), 6.49 (s, 2H), 6.44 (dd, J=8.6, 5.8Hz, 1H), 5.70 (d, J=3.9 Hz, 1H), 5.00-4.92 (m, 1H), 4.90-4.83 (m, 1H),4.33-4.27 (m, 1H), 4.12-3.96 (m, 4H), 3.81 (q, J=12.8, 11.6 Hz, 1H),3.69 (dd, J=12.0, 6.0 Hz, 2H), 2.83-2.77 (m, 1H), 2.65 (d, J=37.6 Hz,12H), 2.23 (dt, J=12.8, 6.7 Hz, 1H), 1.03-0.92 (m, 18H).

Examples 124, 125, and 126:2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10-dihydroxy-2,10-disulfidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one(Diastereomers 1-3)

Step 1:(2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphenyl phosphonate

To a solution of N-(9-((2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide(630 mg, 0.96 mmol) in pyridine (5 mL) under Ar was added diphenylphosphonate (1.07 g, 4.56 mmol), and the mixture was stirred at rt for20 min. It was used for the next reaction step without purification.LCMS (ES, m/z): 798.3 [M+H]⁺.

Step 2: t(2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To the reaction mixture from Step 1 at 0° C. was added water (1 mL),triethylamine (1 mL).

The resulting mixture was stirred at rt for 20 min. Then, it wasconcentrated, and the residue was partitioned between CH₂Cl₂ (50 mL) andaq NaHCO₃ (5%, 20 mL). The organic layer was washed with aq NaHCO₃ (5%,20 mL), dried (Na₂S₂O₄), concentrated and purified by silica gel columnchromatography using 0-7% MeOH in CH₂Cl₂ (1% Et₃N) to give the product.LCMS (ES, m/z): 722.2 [M+H]⁺. ¹H-NMR (400 MHz, CD₃OD): δ 7.76 (s, 1H),7.74 (s, 0.5H), 7.51 (d, J=1.5 Hz, 1H), 7.48 (q, J=2.4, 1.9 Hz, 1H),7.41-7.34 (m, 4H), 7.34-7.27 (m, 2H), 7.26-7.21 (m, 1H), 6.92-6.85 (m,4H), 6.22 (s, 1H), 6.15 (s, 0.5H), 5.37 (d, J=2.7 Hz, 0.5H), 5.28-5.19(m, 1.5H), 4.73-4.69 (m, 0.5H), 4.66-4.62 (m, 1H), 3.80 (s, 6H),3.65-3.55 (m, 1H), 3.53-3.44 (m, 1H), 3.12 (q, J=7.3 Hz, 8H), 2.75 (p,J=6.8 Hz, 1H), 1.33-1.22 (m, 18H). ³¹P-NMR: (162 MHz, CD₃OD): δ 2.67 (s,1P).

Step 3:(2R,3S,4S,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To a stirred solution of(2R,3S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate (540 mg, 0.64 mmol) in CH₂Cl₂ (7 mL) at rt was added water(0.115 g, 6.4 mmol) and 2,2-dichloroacetic acid in CH₂Cl₂ (6%, 7 mL,5.76 mmol). The mixture was stirred at rt for 15 min, and then Et₃SiH(15 mL) was added. After 40 min, pyridine (0.90 mL) was added, and themixture was stirred for 5 min. It was concentrated, and the residue wasused for next reaction step without purification. LCMS (ES, m/z): 419.9[M+H]⁺.

Step 4.(2R,3S,4S,5R)-5-((((((1R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5-dioxabicyclo[2.2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphanyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

The crude from Step 3 was co-evaporated with ACN (3×5 mL), re-dissolvedin ACN (3 mL) under Ar, and dried by adding activated 4 Å molecularsieve (100 mg).(1R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl(2-cyanoethyl) diisopropylphosphoramidite (684.84 mg, 0.774 mmol) wasco-evaporated with ACN (3×5 mL), re-dissolved in ACN (3 mL), and driedby adding activated 4 Å molecular sieve (100 mg). After 30 min, it wasadded to the previously prepared mixture containing pyridin-1-ium (2R,3S,4S,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate. The resulting mixture was stirred at rt for 30 min. Then itwas used in the next reaction step without purification. LCMS (ES, m/z):1202.1 [M+H]⁺.

Step 5.(2R,3S,4S,5R)-5-((((((1R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To the mixture from Step 4 was added(E)-N,N-dimethyl-N′(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide(144.32 mg, 0.704 mmol), and the mixture was stirred at rt for 30 min.Then, it was concentrated, and the crude was used for the next stepwithout purification. LCMS (ES, m/z): 1234.3 [M+H]⁺.

Step 6.(2R,3S,4S,5R)-5-((((((1S,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To a solution of the crude from Step 5 in CH₂Cl₂ (7 mL) at rt was addedwater (120 mg, 6.4 mmol) and 2,2-dichloroacetic acid in CH₂Cl₂ (6%, 7mL, 6 mmol). After 5 min, triethylsilane (15 mL) was added, and it wasstirred for additional 2 h. Then, pyridine (0.9 mL) was added, and itwas concentrated. The residue was purified by reverse phase (C18)chromatography eluted with 0 to 95% ACN in aq NH₄HCO₃ (0.04%) to givethe product. LCMS (ES, m/z): 933.9 [M+H]⁺. ¹H-NMR (400 MHz, CD₃OD): δ8.74-8.64 (m, 1H), 8.58-8.52 (m, 1H), 8.16-8.06 (m, 2H), 7.98-7.88 (m,1H), 7.75 (d, J=4.6 Hz, 0.5H), 7.67 (t, J=7.3 Hz, 1H), 7.60-7.56 (m,2H), 6.25-6.22 (m, 1H), 6.19-6.10 (m, 1H), 5.91 (s, 0.5H), 5.46-5.22 (m,3H), 5.12 (d, J=12.4 Hz, 1H), 4.84-4.49 (m, 3H), 4.35 (tdd, J=13.2, 6.1,3.0 Hz, 2H), 4.14 (d, J=8.5 Hz, 1H), 4.11-3.96 (m, 3H), 2.91 (dt,J=16.4, 6.0 Hz, 2H), 2.81-2.68 (m, 1H), 1.29-1.21 (m, 6H). ³¹P-NMR: (162MHz, CD₃OD): δ 67.84, 66.33 (2 s, 1P); 2.65, 2.52 (2 s, 1P).

Step 7:(5R,7R,8S,12aR,14R,15R,15aS,18S)-2-(2-cyanoethoxy)-18-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10(12H)-olate 2-sulfide

To pyridine (30 mL) at −40° C. under Ar was added diphenylphosphorochloridate (1783.7 mg, 6.64 mmol) and then, a solution of (2R,3S,4S,5R)-5-((((((1S,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate (310 mg, 0.33 mmol, co-evaporated with pyridine 3×5 mL) inCH₂Cl₂ (30 mL) over 20 min. The resulting mixture was stirred at −40° C.for 20 min. It was used in the next step immediately withoutpurification. LCMS (ES, m/z): 916.1 [M+H]⁺.

Step 8.(5R,7R,8S,12aR,14R,15R,15aS,18S)-2-(2-cyanoethoxy)-18-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10(12H)-olate2,10-disulfide

To the solution from Step 7 at −40° C. was added3H-benzo[c][1,2]dithiol-3-one (83.6 mg, 0.498 mmol) and water (179 mg,9.92 mmol). The mixture was stirred at rt for 40 min. Then, it wasconcentrated, and the residue was purified by reverse phase (C18)chromatography eluted with 0 to 95% ACN in aq NH₄HCO₃ (0.04%) to givethe product. LCMS (ES, m/z): 947.8 [M+H]⁺. ¹H-NMR (400 MHz, CD₃OD): δ8.79-8.58 (m, 2H), 8.18-8.06 (m, 2H), 7.92 (d, J=13.5 Hz, 1H), 7.69-7.66(m, 1H), 7.63-7.54 (m, 2H), 6.40-6.14 (m, 2H), 6.03-5.56 (m, 1.5H),5.39-5.10 (m, 2.5H), 4.93-4.85 (m, 2H), 4.84-4.43 (m, 3H), 4.43-3.98 (m,3H), 2.95 (t, J=5.9 Hz, 1H), 2.84-2.66 (m, 2H), 1.30-1.19 (m, 6H).³¹P-NMR (162 MHz, CD₃OD): δ 66.60-64.98 (m, 1P), 56.95-55.65 (m, 1P).

Step 9:2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10-dihydroxy-2,10-disulfidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomers 13)

(5R,7R,8S,12aR,14R,15R,15aS,18S)-2-(2-cyanoethoxy)-18-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10(12H)-olate2,10-disulfide (260 mg, 0.27 mmol) was dissolved in a solution of MeNH₂in EtOH (30%, 20 mL), and the resulting solution was stirred at rt for 3h. Then, it was concentrated, and the residue was purified by Prep-HPLC(Atlantis Prep RP C18 OBD Column, 19 mm×250 mm) eluted with 0 to 14% ACNin aq NH₄HCO₃ (50 mM) over 25 min to afford the diastereomers of2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10-dihydroxy-2,10-disulfidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one:

Example 124 (T_(R): 22.52 min): LCMS (ES, m/z): 719.0 [M−H]⁻. ¹H-NMR:(400 MHz, D₂O+DCl): δ 8.71 (s, 1H), 8.26 (s, 1H), 8.09 (s, 1H), 5.73 (s,1H), 5.65 (s, 1H), 5.22-5.10 (m, 1H), 4.74-4.72 (m, 2H), 4.45 (d, J=4.1Hz, 1H), 4.21-4.11 (m, 1H), 4.05-3.94 (m, 2H), 3.83-3.56 (m, 2H), 3.46(s, 2H). ³¹P-NMR: (162 MHz, D₂O+DCl): δ 60.35 (s, 1P), 56.87 (s, 1P).

Example 125 (T_(R): 15.75 min): LCMS (ES, m/z): 719.0 [M−H]⁻. ¹H-NMR:(400 MHz, D₂O): δ 8.31 (s, 1H), 8.14 (s, 1H), 7.74 (s, 1H), 6.11 (s,1H), 6.06 (s, 1H), 5.61-5.49 (m, 1H), 5.35 (s, 1H), 5.10 (d, J=9.8 Hz,1H), 4.71-4.55 (m, 1H), 4.51-4.20 (m, 3H), 4.18-3.95 (in, 4H). ³P-NMR:(162 MHz, D₂O): δ 54.87-51.81 (m, 2P).

Example 126 (T_(R): 13.17 min): LCMS (ES, m/z): 718.8 [M−H]−. ¹H-NMR:(300 MHz, D₂O+DCl): δ 8.89 (s, 1H), 8.46 (s, 1H), 8.29 (s, 1H), 5.95 (s,1H), 5.88 (s, 1H), 5.30-5.14 (m, 1H), 4.98 (t, J=5.2 Hz, 1H), 4.89 (s,1H), 4.60 (s, 1H), 4.35-4.14 (m, 2H), 4.07 (d, J=11.4 Hz, 1H), 4.01-3.85(m, 2H), 3.68-3.62 (in, 2H). ³P-NM/R: (121 MHz, D₂O+DCl): δ 60.15 (s,1P), 56.60 (s, 1P).

Examples 127 and 128:2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 1), and2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H1-purin-6-one(Diastereomer 2)

Step 1.(2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-ylphenyl phosphonate

To a stirred solution ofN-(3-2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)benzamide(770 mg, 1.138 mmol) in pyridine (5 ml) at 0° C. under Ar was addeddiphenyl phosphonate (1.33 g, 5.69 mmol) over 2 min. The resultingmixture was stirred at rt for 20 min. It was used for the next reactionstep directly without purification.

Step 2:(2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-ylphosphonate

To the reaction mixture from Step 1 at 0° C. was added Et₃N (0.56 mL) inwater (0.56 mL) over 5 min. The resulting mixture was stirred at rt for30 min. It was concentrated, and the residue was partitioned betweenCH₂Cl₂ (60 mL) and aq NaHCO₃ (5%, 24 mL). The organic layer was washedwith aq NaHCO₃ (5%, 2×24 mL), dried (Na₂SO₄), concentrated, and purifiedby chromatography on silica gel using 0-10% MeOH in CH₂Cl₂ (0.5% Et₃N)to give the product. LCMS (ES, m/z): 741.2 [M+H]⁺. ¹H-NMR (400 MHz,CD₃OD): δ 8.79 (s, 1H), 8.21-8.11 (m, 2H), 7.71 (t, J=7.4 Hz, 1H),7.67-7.58 (m, 2.5H), 7.46-7.37 (m, 2H), 7.30-7.23 (m, 4H), 7.18-7.13 (m,2H), 7.03 (dd, J=6.4, 2.8 Hz, 1H), 6.76-6.73 (m, 2H), 6.69-6.65 (m, 2H),6.04 (s, 0.5H), 5.85 (t, J=6.4 Hz, 0.5H), 5.72 (t, J=6.4 Hz, 0.5H), 5.60(td, J=16.9, 6.9 Hz, 1H), 4.44-4.40 (m, 1H), 3.77-3.68 (m, 8H), 3.57(dd, J=10.6, 2.9 Hz, 1H), 2.91 (q, J=7.3 Hz, 18H), 1.20 (t, J=7.3 Hz,27H). ³¹P-NMR: (162 MHz, CD₃OD): δ 2.48 (s, 1P).

Step 3:(2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ylphosphonate

To a solution of(2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-ylphosphonate (610 mg, 0.65 mmol) in CH₂Cl₂ (9.8 mL) was added water (0.12g, 6.5 mmol) and dichloroacetic acid in CH₂Cl₂ (0.6M, 9.8 mL, 5.9 mmol).After 30 in, triethylsilane (20 mL) was added and stirring was continuedfor additional 2 h. Then, pyridine (5 mL) was added, and it wasconcentrated. The crude was used for the next reaction step withoutpurification. LCMS (ES, m/z): 439.1 [M+H]⁺.

Step 4:(2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((((2-cyanoethoxy)(((2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)phosphanyl)oxy)methyl)-4-fluorotetrahydrofuran-3-ylphosphonate

The crude product of Step 3 was co-evaporated with dry ACN (3×3 mL),re-dissolved in ACN (3 mL), and dried by adding activated 4 Å molecularsieve (200 mg). (2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite (0.824 g, 0.960 mmol) wasco-evaporated with dry ACN (3×3 mL), re-dissolved in ACN (5 mL), anddried by adding activated 4 Å molecular sieve (200 mg). After 30 min, itwas added to the previously prepared mixture containing pyridin-1-ium(2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ylphosphonate. It was stirred at rt for 30 min, and the reaction mixturewas used in the next reaction step directly without purification. LCMS(ES, m/z): 893.2 [M+H]⁺.

Step 5: Diastereomers(2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((((2-cyanoethoxy)(((2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-ylphosphonate (116-5-A) and(2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((((2-cyanoethoxy)(((2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-ylphosphonate (116-5-B)

To the reaction mixture from Step 4 at rt, was added(E)-N,N-dimethyl-N′-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide (181mg, 0.880 mmol), and the mixture was stirred for 1 h. Then, it wasconcentrated, and the residue was purified by reverse phase (AQ-C18)chromatography eluted with 0 to 28% ACN in aq NH₄HCO₃ (5 mM) over 88min.

The first fractions with desired mass (T_(R)=50 min) gave(2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((((2-cyanoethoxy)(((2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-ylphosphonate (116-5-A). LCMS (ES, m/z): 924.9 [M+H]⁺. ¹H-NMR: (400 MHz,CD₃OD): δ 8.90 (s, 1H), 8.31 (s, 1H), 8.10 (d, J=7.8 Hz, 2H), 7.75 (s,0.5H), 7.69 (t, J=7.4 Hz, 1H), 7.57 (t, J=7.7 Hz, 2H), 7.02 (dd, J=6.5,2.6 Hz, 1H), 6.27-6.13 (m, 1.5H), 5.79-5.51 (m, 3H), 5.47-5.21 (m, 12H),4.58 (dd, J=15.4, 7.6 Hz, 2H), 4.50-4.25 (m, 3H), 4.05 (ddd, J=24.7,13.2, 6.1 Hz, 2H), 3.90-3.76 (m, 4H), 2.85-2.56 (m, 3H), 1.34 (d, J=6.5Hz, 3H), 1.25-1.19 (m, 3H). ³¹P-NMR (162 MHz, CD₃OD): δ 67.56 (s, 1P),3.09 (s, 1P).

The second fractions with desired mass (T_(R)=55 min) gave(2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((((2-cyanoethoxy)(((2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-ylphosphonate (116-5-B). LCMS (ES, m/z): 925.1 [M+H]⁺. ¹H-NMR: (400 MHz,CD₃OD): δ 8.87 (s, 1H), 8.33 (s, 1H), 8.16 (d, J=7.8 Hz, 2H), 7.75 (s,0.5H), 7.70 (t, J=7.4 Hz, 1H), 7.61 (t, J=7.7 Hz, 2H), 6.91 (dd, J=6.2,3.4 Hz, 1H), 6.23 (d, J=7.1 Hz, 1H), 6.17 (s, 0.5H), 5.74-5.54 (m, 3H),5.46 (d, J=4.6 Hz, 0.5H), 5.33 (d, J=4.6 Hz, 0.5H), 4.65-4.24 (m, 4H),4.15 (dt, J=13.0, 5.8 Hz, 2H), 3.95-3.75 (m, 2H), 2.90-2.67 (m, 3H),1.36-1.31 (m, 3H), 1.19 (d, J=6.8 Hz, 3H). ³¹P-NMR: (162 MHz, CD₃OD): δ67.29 (s, 1P), 3.07 (s, 1P).

Step 6:(5R,7R,8S,12aR,14R,15S,15aR,16R)-10-(2-cyanoethoxy)-15,16-difluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{7-[(phenylcarbonyl)amino]-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-2-olate10-sulfide

To pyridine (10 mL) at −40° C. under Ar was added diphenylphosphorochloridate (628 mg, 2.34 mmol), and then, a solution of(2R,3R,4S,5R)-5-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2-((((2-cyanoethoxy)(((2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)phosphorothioyl)-oxy)methyl)-4-fluorotetrahydrofuran-3-ylphosphonate (116-5-B) (110 mg, 0.105 mmol, co-evaporated with pyridine3×5 mL) in CH₂Cl₂ (10 mL) over 20 min. It was stirred at −40° C. for 20min. The reaction mixture was used in the next step immediately withoutpurification.

Step 7:(5R,7R,8S,12aR,14R,15S,15aR,16R)-10-(2-cyanoethoxy)-15,16-difluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{7-[(phenylcarbonyl)amino]-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-2-olate2,10-disulfide

To the mixture from Step 6 at −20° C. was added3H-benzo[c][1,2]dithiol-3-one (27.8 mg, 0.165 mmol) and water (42 mg,2.3 mmol). The resulting mixture was stirred at rt for 30 min. It wasconcentrated, and the residue was purified by reverse phase (AQ-C18)chromatography eluted with 0 to 28% ACN in aq NH₄HCO₃ (5 mM) to give theproduct. LCMS (ES, m/z): 938.9 [M+H]⁺. ¹H-NMR: (400 MHz, CD₃OD): δ8.94-8.71 (m, 2H), 8.23-8.06 (m, 2H), 7.75-7.67 (m, 1H), 7.64 (t, J=7.5Hz, 2H), 7.11-6.97 (m, 1H), 6.32-6.17 (m, 2H), 6.02-5.73 (m, 2H),5.68-5.38 (m, 1H), 4.73 (d, J=24.3 Hz, 1H), 4.68-4.42 (m, 4H), 4.39-4.19(m, 2H), 4.06-3.66 (m, 2H), 2.99-2.62 (m, 2H), 2.61-2.37 (m, 1H), 1.20(d, J=6.8 Hz, 3H), 1.12 (d, J=6.7 Hz, 3H). ³¹P-NMR: (162 MHz, CD₃OD): δ64.05, 63.79 (2 s, 1P); 56.67, 56.27 (2 s, 1P).

Step 8: 2-amino-9-(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomers 1 and 2)

To pyridinium(5R,7R,8S,12aR,14R,15S,15aR,16R)-10-(2-cyanoethoxy)-15,16-difluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{7-[(phenylcarbonyl)amino]-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-2-olate2,10-disulfide in a steel tank (150 mL) at −60° C. was added ammonia inisopropanol (50 mL) at −60° C. The reactor was tightly sealed and then,heated at 50° C. for 16 h. Then, the volatile components were removedunder reduced pressure, and the residue was purified by prep-HPLC(Atlantis Prep T3 OBD Column, 19×250 mm) eluted with 0 to 5% ACN in aqNH₄HCO₃ (50 mM) over 25 min.

Example 127 (T_(R)=17.32 min):2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 1). LCMS (ES, m/z): 709.8 [M−H]⁻. ¹H-NMR: (400 MHz, D₂O):δ 8.32 (s, 1H), 8.27 (s, 1H), 6.78 (dd, J=11.1, 4.9 Hz, 1H), 6.07 (d,J=8.6 Hz, 1H), 5.81 (t, J=4.6 Hz, 0.5H), 5.68 (t, J=4.7 Hz, 0.5H), 5.63(dq, J=9.8, 5.0 Hz, 1H), 5.58 (d, J=3.5 Hz, 0.5H), 5.45 (d, J=3.4 Hz,0.5H), 5.43-5.26 (m, 1H), 4.65-4.63 (m, 1H), 4.56 (q, J=5.6 Hz, 1H),4.31-4.23 (m, 3H), 4.12-4.05 (m, 1H). ³¹P-NMR: (162 MHz, D₂O): δ 55.76(s, 1P), 54.26 (s, 1P).

Example 128 (T_(R)=21.10 min):2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 2). LCMS (ES, m/z): 709.8 [M−H]⁻. ¹H-NMR: (400 MHz, D₂O):δ 8.26 (s, 1H), 8.01 (s, 1H), 6.79 (dd, J=9.4, 5.3 Hz, 1H), 6.05 (d,J=8.6 Hz, 1H), 5.85 (t, J=5.0 Hz, 0.5H), 5.72 (t, J=5.0 Hz, 0.5H),5.65-5.35 (m, 3H), 4.69-4.62 (m, 1H), 4.48 (d, J=5.5 Hz, 1H), 4.42 (t,J=11.1 Hz, 1H), 4.18 (t, J=6.2 Hz, 2H), 4.09-4.06 (m, 1H). ³¹P-NMR (162MHz, D₂O): δ 54.74 (s, 1P), 53.84 (s, 1P).

Examples 129 through 243, as shown in Table 6 below, were or may beprepared according to procedures analogous to those outlined in Examples116 through 128 above using the appropriate monomers, described asPreparations or as obtained from commercial sources, in the couplingstep.

TABLE 6 Mass Ex. Structure Name [M − H]⁻ 129

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetra-decin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3) 710 130

2-amino-9- [(2R,5S,7R,8R,10R,12aR,14R,15S,15aR)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 1) 691 131

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 2) 691 132

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-oe(Diastereomer 3) 691 133

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 694 134

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aS,18R)-14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10-dioxido-2,10-disulfanylhexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 719 135

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aS,18R)-14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10-dioxido-2,10-disulfanylhexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7(12H)-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 2)719 136

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aS,18R)-14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10-dioxido-2,10-disulfanylhexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3) 719 137

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aS,18R)-14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10-dioxido-2,10-disulfanylhexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 4) 719 138

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15-fluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one 692 139

2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(6-amino-9H-purin-9-yl)-2,10-dioxido-2,10-disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 701 140

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)-14-(6-amino-9H-purin-9-yl)-2,10-dioxido-2,10-disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 701 141

2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(6-amino-9H-purin-9-yl)-2,10-dioxido-2,10-disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one (Diasteroemer 3) 701 142

2-amino-9-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(6-amino-9H-purin-9-yl)-2,10-dioxido-2,10-disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 4) 701 143

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-10-oxido-2-sulfidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one 694 144

2-amino-9- [(2R,5R,7R,8S,10R,12aR,14R,15S,15aR,16S)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 1) 709 145

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 2) 709 146

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 3) 709 147

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetra-decin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 710 148

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetra-decin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 710 149

2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 1) 673 150

2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 2) 673 151

2-amino-9-[(2R,5S,7R,8R,10R, 12aR,14R,15aS)-14-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 3) 673 152

2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 4) 673 153

(5R,7R,8R,12aR,14R,15S,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-15,16-difluorooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecine-2,10-bis(thiolate) 2,10- dioxide (Diastereomer 1) 725154

(5R,7R,8R,12aR,14R,15S,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-15,16-difluorooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecine-2,10-bis(thiolate) 2,10-dioxide (Diastereomer 2) 725155

2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR)-14-(6-amino-9H-purin-9-yl)-15,16,16-trifluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 727 156

2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR)-14-(6-amino-9H-purin-9-yl)-15,16,16-trifluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 727 157

2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aS)-14-(6-amino-9H-purin-9-yl)-18,18-difluoro-2,10-dihydroxy-2,10-disulfidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7(12H)-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 1)737 158

2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aS)-14-(6-amino-9H-purin-9-yl)-18,18-difluoro-2,10-dihydroxy-2,10-disulfidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7(12H)-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 2)737 159

2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aS)-14-(6-amino-9H-purin-9-yl)-18,18-difluoro-2,10-dihydroxy-2,10-disulfidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7(12H)-yl]-1,9-dihydro-6H- purin-6-one (Diastereomer 3)737 160

2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aS)-14-(6-amino-9H-purin-9-yl)-18,18-difluoro-2,10-dihydroxy-2,10-disulfidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7(12H)-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 4)737 161

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 690 162

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 690 163

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-1,9-dihydro-6H- purin-6-one (Diastereomer 3) 690164

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 4) 690 165

2-amino-9-[(5R,7R,8S,12aR,14R,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,15,16-trifluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 728 166

2-amino-9-[(5R,7R,8S,12aR,14R,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,15,16-trifluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 728 167

2-amino-9-[(5R,7R,8S,12aR,14R,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,15,16-trifluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3) 728 168

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 708 169

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 708 170

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3) 708 171

2-amino-9-[(2R,5S,7R,8R,10R, 12aR,14R,15S,15aR)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 690 172

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 690 173

2-amino-9-[(2R,5R,7R,8S,10R, 12aR,14R,15S,15aR,16S)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H- 5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 708 174

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 708 175

5-amino-3-[(5S,7R,8R,12aR,14R,15S,15aR)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 1) 692 176

5-amino-3-[(5S,7R,8R,12aR,14R,15S,15aR)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 2) 692 177

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d] pyrimidin-7-one (Diastereomer 1) 710 178

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 2) 710 179

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 3) 710 180

1-[(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-1,5-dihydro-4H- imidazo[4,5-c]pyridin-4-one 693181

5-amino-3-[(5S,7R,8R,12aR,14R,15S,15aR)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 1) 693 182

5-amino-3-[(5S,7R,8R,12aR,14R,15S,15aR)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo-tetradecin-7-yl]-3,6-dihydro-7H-[1,2,3] triazolo[4,5-d]pyrimidin-7-one(Diastereomer 2) 693 183

5-amino-3-[(5S,7R,8R,12aR,14R,15S,15aR)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 3) 693 184

5-amino-3-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(6-amino-9H-purin-9-yl)-2,10-dihydroxy-2,10-disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7(12H)-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 1) 702 185

5-amino-3-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(6-amino-9H-purin-9-yl)-2,10-dihydroxy-2,10-disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7(12H)-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 2) 702 186

5-amino-3-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(6-amino-9H-purin-9-yl)-2,10-dihydroxy-2,10-disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7(12H)-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d] pyrimidin-7-one (Diastereomer 3) 702187

5-amino-3-[(5S,7R,8R,12aR,14R,15R,15aS)-14-(6-amino-9H-purin-9-yl)-2,10-dihydroxy-2,10-disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7(12H)-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 4) 702 188

5-amino-3-[(5S,7R,8R,12aR,14R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 1) 673 189

5-amino-3-[(5S,7R,8R,12aR,14R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 2) 673 190

5-amino-3-[(5S,7R,8R,12aR,14R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 3) 673 191

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 708192

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 708193

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one 706 194

5-amino-3-[(5S,7R,8R,12aR,14R,15S,15aR)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 1) 691 195

5-amino-3-[(5S,7R,8R,12aR,14R,15S,15aR)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 2) 691 196

5-amino-3-[(5S,7R,8R,12aR,14R,15S,15aR)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 3) 691 197

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 1) 710 198

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 2) 710 199

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 3) 710 200

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d] pyrimidin-3-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 1) 711 201

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 2) 711 202

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one 711 203

5-amino-3- [(5R,7R,8S,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10-dioxido-2,10-disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7(12H)-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7- one (Diastereomer 1) 720 204

5-amino-3- [(5R,7R,8S,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10-dioxido-2,10-disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7(12H)-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 2) 720 205

5-amino-3- [(5R,7R,8S,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10-dioxido-2,10-disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7(12H)-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 3) 720 206

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15,16-difluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d] pyrimidin-7-one 709 207

5-amino-3-[(5R,7R,8S,112aR,14R,15aS,16S)-14-(6-amino-9H-purin-9-yl)-12a-ethynyl-16-fluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 1) 716 208

5-amino-3-[(5R,7R,8S,112aR,14R,15aS,16S)-14-(6-amino-9H-purin-9-yl)-12a-ethynyl-16-fluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 2) 716 209

5-amino-3-[(5R,7R,8S,112aR,14R,15aS,16S)-14-(6-amino-9H-purin-9-yl)-12a-ethynyl-16-fluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclo-tetradecin-7-yl]-3,6-dihydro-7H-[1,2,3] triazolo[4,5-d]pyrimidin-7-one(Diastereomer 3) 716 210

5-amino-3-[(5R,7R,8S,12aR,14R,15aS,16S)-14-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-16-fluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 1) 692 211

5-amino-3-[(5R,7R,8S,12aR,14R,15aS,16S)-14-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-16-fluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 2) 692 212

5-amino-3-[(5R,7R,8S,12aR,14R,15aS,16S)-14-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 1) 692 213

5-amino-3-[(5R,7R,8S,12aR,14R,15aS,16S)-14-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 2) 692 214

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-1,9-dihydro-6H- purin-6-one (Diastereomer 1) 725215

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-1,9-dihydro-6H- purin-6-one (Diastereomer 2) 725216

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-1,9-dihydro-6H- purin-6-one (Diastereomer 3) 725217

2-amino-9- [(2R,5R,7R,8R,10R,12aR,14R,15S,15aR,16S)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-1,9-dihydro-6H- purin-6-one (Diastereomer 4) 725218

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-1,9-dihydro-6H- purin-6-one (Diastereomer 1) 726219

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 726220

5-amino-3-[(5R,7R,8S,12aR,14R,15aR,16S)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15,15,16-trifluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one 727 221

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-3,6-dihydro- 7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 1) 726 222

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-3,6-dihydro- 7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 2) 726 223

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 3) 726 224

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d] pyrimidin-3-yl)-15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7- yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin- 7-one (Diastereomer 1) 727 225

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 2) 727 226

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 1) 725 227

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-3,6-dihydro-7H- [1,2,3]triazolo[4,5-d]pyrimidin-7-one (Diastereomer 2) 725 228

1-[(5S,7R,8R,12aR,14R,15S,15aR)- 14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15-fluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,5-dihydro-4H-imidazo[4,5-c]pyridin-4-one 674 229

6-amino-1- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha- cyclotetradecin-7-yl]-1,5-dihydro-4H-imidazo[4,5-c]pyridin-4-one (Diastereomer 1) 707 230

6-amino-1- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha-cyclotetradecin-7-yl]-1,5-dihydro-4H- imidazo[4,5-c]pyridin-4-one(Diastereomer 2) 707 231

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one(Diastereomer 1) 726 232

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 726233

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3) 726234

2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15-fluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one 706 235

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]- 3,6-dihydro-7H-[1,2,3]triazolo[4,5-d]pyrimidin-7-one 726 236

2-amino-9-[(5R,7R,8R,12aR,14R,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,15,16-trifluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 744 237

2-amino-9-[(5R,7R,8R,12aR,14R,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,15,16-trifluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-1,9-dihydro-6H- purin-6-one (Diastereomer 2) 744238

2-amino-9-[(5R,7R,8R,12aR,14R,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,15,16-trifluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3) 744239

2-amino-9-[(5R,7R,8R,12aR,14R,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,15,16-trifluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7-yl]-1,9-dihydro-6H- purin-6-one (Diastereomer 4) 744240

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10-dioxido-2,10-disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7(12H)-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 1)735 241

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10-dioxido-2,10-disulfanylhexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha- cyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 735 242

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10-dioxido-2,10-disulfanylhexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphospha-cyclotetradecin-7(12H)-yl]-1,9- dihydro-6H-purin-6-one (Diastereomer 3)735 243

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-imidazo[4,5-b]pyridin-3-yl)- 15,16-difluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7- yl]-1,9-dihydro-6H-purin-6-one724

Examples 244, 245, 246, and 247:2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,1-dihydroxy-2,1-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomers 1-3) and2-amino-9-1(2R,5R,7R,8S,10R,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 4)

Step 1.(2R,3R,4R,5R)-5-((((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphanyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylhydrogen phosphonate

Pyrrole (0.087 mL, 1.2 mmol) was added to a solution of(2R,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylhydrogen phosphonate triethylamine salt (1:2) (0.34 g, 0.41 mmol) inacetonitrile (3.0 mL) under an argon atmosphere at 0° C. After 5 min,TFA (0.096 mL, 0.14 mmol) was added, and the reaction mixture wasstirred at 0° C. for 30 min. Pyridine (0.13 mL, 1.7 mmol) was added dropwise at 0° C. The reaction mixture was then stirred for 10 min at 0° C.At that time, a mixture of(2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite (0.48 g, 0.55 mmol) inacetonitrile (3.0 mL) was added drop wise over 5 min to the reactionmixture under an argon atmosphere at 0° C. The reaction mixture wasstirred at 0° C. for 20 min and immediately used in the next stepwithout further manipulation.

Step 2:(2R,3S,4R,5R)-5-((((((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylhydrogen phosphonate

To the crude reaction mixture from Step 1 was added(E)-N,N-dimethyl-N′-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide(0.10 g, 0.50 mmol) under an argon atmosphere at 0° C. The reactionmixture was stirred for 45 minutes at 0° C. At that time, 1-propanol(0.31 mL, 4.13 mmol) was added to the reaction mixture under an argonatmosphere at 0° C. The reaction mixture was then allowed to warm toambient temperature and stirred for 10 min. TFA (0.32 mL, 4.1 mmol) wasadded to the reaction mixture, and the reaction mixture was stirred for30 min at ambient temperature. Pyridine (0.37 mL, 4.6 mmol) was added atambient temperature, and the reaction mixture was stirred for 10 min.The reaction mixture was concentrated under reduced pressure toapproximately one-half volume. The mixture was then diluted withisopropyl acetate (20 mL) and stirred for 30 min at ambient temperature.The resulting suspension was filtered.

The collected solids were dried overnight under high vacuum to afford(2R,3S,4R,5R)-5-((((((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylhydrogen phosphonate. LCMS (ES, m/z): 922 [M−H]⁻.

Step 3:2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

(2R,3S,4R,5R)-5-((((((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)-methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylhydrogen phosphonate (0.30 g, 0.33 mmol) was azeotroped with drypyridine (2×10 mL) and then dried under high vacuum for 1 h. In aseparate flask, diphenyl phosphorochloridate (0.34 mL, 1.6 mmol) wasadded to a mixture of acetonitrile (15 mL) and pyridine (1.0 mL). Theresulting solution was then cooled to −20° C. To this mixture was addeddrop wise over a period of 5 min a mixture of (2R,3S,4R,5R)-5-((((((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)-methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylhydrogen phosphonate (0.30 g, 0.33 mmol) in pyridine (4.0 mL) at −20° C.The reaction mixture was then stirred at −20° C. for 15 minpost-addition. 3H-benzo[c][1,2]dithiol-3-one (0.066 g, 0.39 mmol) andwater (0.12 mL, 6.5 mmol) were then added to the reaction mixture at−20° C. The reaction mixture was allowed to gradually warm to ambienttemperature. The reaction mixture was stirred for 30 min at ambienttemperature. The reaction mixture was then concentrated under reducedpressure to approximately one quarter volume. The reaction mixture wascooled to 0° C., and methanamine (33% in ethanol) (2.63 mL, 24 mmol) wasadded drop wise. After the addition was complete, the reaction mixturewas allowed to warm to ambient temperature. The reaction mixture wasstirred at ambient temperature for 18 h. The reaction mixture wasconcentrated under reduced pressure to afford the crude product residue.The crude product residue was azeotroped (3×30 mL ethanol) to afford thecrude product. This material was dissolved in water (5 mL) andacetonitrile (1 mL). The resulting mixture was purified by mass-directedreverse phase HPLC (Waters Sunfire 19×250 mm, UV 215/254 nm, fractiontrigger by SIM negative MS monitoring mass 709; mobile phase=100 mMtriethylammonium acetate in water/acetonitrile gradient, 2-30%acetonitrile over 40 min) to afford the 4 diastereomers of2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one.

Example 244:2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 1): T_(R)=17.7 min. LCMS (ES, m/z): 709 [M−H]⁻.

Example 245:2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 2): T_(R)=21.9 min. LCMS (ES, m/z): 709 [M−H]⁻. 1H NMR(500 MHz, DMSO-d₆) δ 8.32 (s, 1H), 8.21-8.09 (m, 2H), 7.46-7.29 (m, 2H),6.59-6.43 (m, 2H), 6.40-6.29 (m, 1H), 5.88 (d, J=8.8 Hz, 1H), 5.49-5.19(m, 4H), 4.45-4.32 (m, 2H), 4.10-3.93 (m, 2H), 3.94-3.82 (m, 1H),3.80-3.68 (m, 1H).

Example 246:2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 3): T_(R)=23.8 min. LCMS (ES, m/z): 709 [M−H]⁻. 1H NMR(500 MHz, DMSO-d₆) δ 8.18-8.08 (m, 3H), 7.41-7.33 (m, 2H), 6.59-6.47 (m,2H), 6.37-6.27 (m, 1H), 5.84 (d, J=8.7 Hz, 1H), 5.52-5.26 (m, 2H),5.21-5.11 (m, 1H), 4.46-4.35 (m, 2H), 4.19-4.02 (m, 2H), 3.83-3.65 (m,2H).

Example 247: 2-amino-9-[(2R,5R,7R,8S,10R,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 4): T_(R)=26.4 min. LCMS (ES, m/z): 709 [M−H]⁻. ¹H NMR(500 MHz, DMSO-d₆) δ 8.19-8.07 (m, 3H), 7.41-7.32 (m, 2H), 6.70-6.50 (m,2H), 6.40-6.29 (m, 1H), 5.85 (d, J=8.7 Hz, 1H), 5.33-5.25 (m, 2H),5.23-5.12 (m, 1H), 4.48-4.35 (m, 1H), 4.33-4.24 (m, 1H), 4.09-3.93 (m,2H), 3.92-3.81 (m, 1H), 3.83-3.70 (m, 1H).

Examples 248 through 256, as shown in Table 7 below, were preparedaccording to procedures analogous to those outlined in Examples 244through 247 above using the appropriate monomers, described asPreparations or as obtained from commercial sources, in the couplingstep.

TABLE 7 Mass Ex. Structure Name [M − H]⁻ 248

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-16-fluoro-2,10-dioxido-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 691 249

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-16-fluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 691 250

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-16-fluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3) 691 251

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-aminoimidazo[2,1-f)[1,2,4]triazin-7-yl)-16-fluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 4) 691 252

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-fluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 708 253

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-fluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 708 254

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-fluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3) 708 255

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-amino-5-fliioro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-fluoro-10-hydroxy-2,10-dioxido-2-sulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 692 256

2-amino-9-[(5R,7R,8S,12aR,4R,15aS,16R)-14-(4-amino-5-fliioro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-fluoro-10-hydroxy-2,10-dioxido-2-sulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 692

Examples 257 through 259 in Table 8 were made according to proceduresanalogous to those described above for Examples 244 through 247 usingthe appropriate monomeric nucleosides, described as Preparations or asobtained from commercial sources, with the following additionalrepresentative treatment as a final step: A sample (0.12 mmol) wasdissolved in water (6 mL), and the resulting mixture was applied to ionexchange resin in a column (DOWEX 50WX2 hydrogen form, 100-200 mesh, 1.5g, pre-washed with 10 mL water, and then packed in column beforecompound loading occurred). After loading mixture had completelyabsorbed to column, the column was then washed with additional water (10mL). The eluent was lyophilized to afford product.

TABLE 8 Mass Ex. Structure Name [M − H]⁻ 257

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 691 258

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 691 259

2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 3) 691

Alternatively, Examples 117-142, 144-152, 172, 174, and 244-247, abovewere made using procedures similar to those described for Examples 81through 83 above.

Example 260:2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

Step 1.((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3-((triethylsilyl)oxy)tetrahydrofuran-2-yl)methyl(2-cyanoethyl) diisopropylphosphoramidite

To a solution ofN-(9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (780 mg, 2.089 mmol) inDMF (8 mL) and DIPEA (1.116 mL, 6.39 mmol) at 0° C. was added 200 mgactive molecular sieve 4 Å and 2-cyanoethylN,N-diisopropylchlorophosphoramidite (572 mg, 2.343 mmol) in 1 ml dryCH₃CN. The resulting mixture was stirred at 0° C. for 5 h;chlorotriethylsilane (401 mg, 2.66 mmol) was added dropwise. Theresulting mixture was stirred at rt overnight. The reaction carrieddirectly to next step. LCMS (ES, m/z): 686 [M−H]⁻

Step 2:O-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3-((triethylsilyl)oxy)tetrahydrofuran-2-yl)methyl)O-((2R,3S,4R,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)O-(2-cyanoethyl)phosphorothioate

To a solution of the product of step 1 was added N-(9-((2R,3S,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide(500 mg, 1.065 mmol) and 1H-tetrazole (895 mg, 12.78 mmol). The mixturewas stirred at RT for 2 h, and DDTT (568 g, 2.77 mmol) was added. Themixture was stirred for 1 h, then partitioned between ethyl acetate andH₂O, dried over Na₂SO₄, filtered and concentrated in vacuo. The residuewas purified by column chromatography on silica gel, eluting with 0-6%MeOH/DCM to giveO-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3-((triethylsilyl)oxy)tetrahydrofuran-2-yl)methyl)O-((2R,3S,4R,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)O-(2-cyanoethyl)phosphorothioate. LCMS (ES, m/z): 1088 [M+H]⁺

Step 3:O-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl)O-((2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl)O-hydrogenphosphorothioate

To a solution of the mixture (520 mg) from step 3 in THF (5 mL) wasadded TBAF (1.0 M in THF) (1.140 mL, 1.140 mmol). The resulting mixturewas stirred at RT for 2 h and concentrated in vacuo. The residue waspurified by column chromatography on silica gel, eluting with 0-8%MeOH/DCM to giveO-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl)O-((2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl)O-hydrogenphosphorothioate. LCMS (ES, m/z): 807 [M+H]⁺

Step 4.N-{9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-15,16-difluoro-2,10-dihydroxy-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}benzamide

O-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl)O-((2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl)O-hydrogenphosphorothioate (250 mg, 0.310 mmol) and diisopropylammoniumtetrazolide (80 mg, 0.465 mmol) were azeotrope with dry CH₃CN (3×10 ml)and dried under high vacuum for 30 min.

The above mixture was dissolved in DMF (1 mL) and acetonitrile (7 mL)and added 200 mg active molecular sieve 4 Å and a solution of2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (128 mg, 0.403mmol) in 1 ml dry CH₃CN. The resulting mixture was stirred at rt for 30min, followed by addition of 1H-tetrazole (109 mg, 1.550 mmol). Thereaction stayed at rt for 1 h and added DDTT (95 mg, 0.465 mmol). Thestirring continued for 1 h and concentrated in vacuo. The residue waspurified by column chromatography on silica gel, eluting with 0-10%MeOH/DCM with 1% ET3N to give the desired product. LCMS (ES, m/z): 883[M−H]⁻.

Step 5:2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

N-{9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-15,16-difluoro-2,10-dihydroxy-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}benzamide(120 mg, 0.136 mmol) and ammonia (7.0M in MeOH) (2 ml, 14.00 mmol) weresealed in a microwave tube. The reaction mixture was heated to 50° C.and stirred for 4 h. The reaction mixture was concentrated, and purifiedusing mass-directed reverse phase HPLC (X-Bridge BEH 150 Prep C18) usinga gradient solvent system with MeCN and 100 mM aqueous triethylammoniumacetate to give desired product. Lyopholization of the product fractionsfurnished2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]-pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one.LCMS (ES, m/z): 709 [M−H]⁻. ¹H NMR (H₂O-d2, 500 MHz): δ_(H) 8.23 (1H,s), 8.17 (1H, s), 7.93 (1H, s), 6.43 (1H, d, J=15.0 Hz), 6.00 (1H, d,J=8.7 Hz), 5.75 (1H, m), 5.58 (2H, m), 5.15 (1H, m), 4.65 (2H, m), 4.54(1H, m), 4.15-4.30 (3H, m). ³¹P NMR: (H₂O-d₂, 202 MHz): δ 52.1, 52.2.

Examples 261, 262, 263:2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-12a-ethynyl-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomers 1-3)

Step 1:((2R,3R,4S,5R)-4-(((2-cyanoethoxy)(diisopropylamino)phosphanyl)oxy)-3-fluoro-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl(2-cyanoethyl) diisopropylphosphoramidite

To a solution ofN-(9-((2R,3R,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (190mg, 0.535 mmol) and3-((bis(diisopropylamino)phosphino)oxy)propanenitrile (95%) (373 mg,1.176 mmol) in DMF (5 ml) was added diisopropylammonium tetrazolide (137mg, 0.802 mmol). The mixture was stirred at RT for 2 h, then added1H-tetrazole (15.77 mg, 0.225 mmol) and continued for 2 h. 300 mg activemolecular sieve 4 Å was added and continued stirring at RT for 2 h. Thereaction carried directly to next step. LCMS (ES, m/z): 754 [M−H]⁻

Step 2:N-{7-[(5R,7R,8S,12aR,14R,15aS,16R)-2,10-bis(2-cyanoethoxy)-12a-ethynyl-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-4-yl}benzamide

To a solution of N-(7-((2R,4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide(180 mg, 0.454 mmol) and Et₃N (0.114 mL, 0.817 mmol) in DMF (4 mL) at−78° C. was added a solution of TMS-Cl (0.070 mL, 0.545 mmol) in CH₂Cl₂(1 mL) dropwise. The mixture was stirred at −78° C. for 20 min andwarmed to 0° C. within 1 h, then added 200 mg active molecular sieve 4 Åand continued stirring at RT for 2 h. The mixture was cooled to 0° C.and transferred to a stirred solution of the product of Step 1 (394 mg,0.454 mmol) (precooled to −0° C.), followed by addition of 1H-tetrazole(191 mg, 2.72 mmol). The mixture was gradually warmed to RT and stirredat RT for 2 h. The filtration removed the solids. After washed with 3 mlDMF, the combined filtrate was added additional 1H-tetrazole (191 mg,2.72 mmol) and stirred at RT overnight.

After completion of reaction, the reaction mixture was diluted withethyl acetate and washed successively with H₂O and brine. The organiclayer was dried over anhydrous Na₂SO₄ and purified by a silica gelchromatography, eluting with 0-8% MeOH/DCM to give two desired fractionswith the same molecular weight. LCMS (ES, m/z): 1014 [M+H]⁺

Step 3:2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-12a-ethynyl-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

N-{7-[(5R,7R,8S,12aR,14R,15aS,16R)-2,10-bis(2-cyanoethoxy)-12a-ethynyl-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-4-yl}benzamide(faster fraction from step 2) (30 mg, 0.030 mmol) and 7.0M ammonia inMeOH (2 ml, 14.00 mmol) were sealed in a microwave tube. The mixture washeated to 50° C. and stirred for 8 h. The reaction mixture wasconcentrated, and the product was purified using mass-directed reversephase HPLC (X-Bridge BEH 150 Prep C18) using a gradient solvent systemwith MeCN and 100 mM aqueous triethylammonium acetate. Lyopholization ofthe product fractions furnished2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-12a-ethynyl-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-oneas the bis-triethylamine salt.

Example 261 (Diastereomer 1): LCMS (ES, m/z): 732 [M−H]⁻. ¹H NMR(H₂O-d₂, 500 MHz): δ_(H) 8.32 (1H, s), 8.08 (1H, s), 7.05 (1H, s), 6.70(1H, t, J=6.1 Hz), 6.08 (1H, d, J=8.7 Hz), 5.71 (1H, d, J=53.2 Hz),5.47-5.40 (1H, m), 5.14-5.10 (1H, m), 4.65 (2H, m), 4.28 (1H, t, J=8.7Hz), 4.14-4.02 (3H, m), 2.85 (2H, d, J=6.4 Hz). ³¹P NMR: (H₂O-d₂, 202MHz): δ 53.7, 54.0.

Step 4:2-amino-9-[(5R,7R,8S,12aR,14R,15aS,16R)-14-(4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-12a-ethynyl-16-fluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

N-{7-[(5R,7R,8S,12aR,14R,15aS,16R)-2,10-bis(2-cyanoethoxy)-12a-ethynyl-16-fluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-4-yl}benzamide(slow fraction from Step 2, 65 mg, 0.064 mmol) and 7.0 M ammonia in MeOH(2 ml, 14.00 mmol) were sealed in a microwave tube and heated to 50° C.and stirred for 8 h. The reaction mixture was concentrated and purifiedusing mass-directed reverse phase HPLC (X-Bridge BEH 150 Prep C18) usinga gradient solvent system with MeCN and 100 mM aqueous triethylammoniumacetate to give two additional diastereomers after lyopholization of theproduct fractions.

Example 262 (Diastereomer 2): LCMS (ES, m/z): 732 [M−H]⁻. ¹H NMR(H₂O-d₂, 500 MHz): δ_(H) 8.09 (2H, d, J=3.0 Hz), 7.19 (1H, s), 6.74 (1H,s), 6.08 (1H, d, J=8.3 Hz), 5.63 (1H, s), 5.53 (1H, s), 5.35-5.33 (1H,m), 4.65 (2H, m), 4.34 (1H, d, J=8.8 Hz), 4.28 (1H, t, J=10.3 Hz), 4.11(1H, d, J=12.1 Hz), 3.89 (1H, dd, J=10.6, 4.0 Hz), 2.85 (2H, d, J=6.4Hz). ³¹P NMR: (H₂O-d₂, 202 MHz): δ 54.7, 59.8.

Example 263 (Diastereomer 3): LCMS (ES, m/z): 732 [M−H]⁻. ¹H NMR(H₂O-d₂, 500 MHz): δ_(H) 8.08 (1H, s), 7.94 (1H, s), 7.04 (1H, s), 6.68(1H, s), 6.04 (1H, d, J=8.6 Hz), 5.64 (1H, s), 5.54 (1H, d, J=3.3 Hz),5.24 (1H, d, J=7.7 Hz), 4.65 (2H, m), 4.40 (1H, d, J=8.2 Hz), 4.18 (1H,dd, J=11.0, 5.5 Hz), 4.09-4.00 (2H, m), 2.93-2.89 (1H, m), 2.82 (1H, d,J=7.1 Hz). ³¹P NMR: (H₂O-d₂, 202 MHz): δ 53.1, 54.9.

Examples 264 and 265:2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-2,10,18-trihydroxy-2-oxido-10-sulfidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 1) and2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-2,10,18-trihydroxy-2-oxido-10-sulfidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 2)

Step 1:(2R,3R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-ylphosphonate

To a solution of(2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-ylphosphonate (750 mg, 0.865 mmol) in CH₂Cl₂ (9 mL) was added water (156mg, 8.65 mmol) and 2,2-dichloroacetic acid in CH₂Cl₂ (0.6M, 11 mL, 6.6mmol). The mixture was stirred at rt for 15 min, and then, Et₃SiH (4.00mL) was added. After 1 h, pyridine (1232 mg, 15.57 mmol) was added, andit was concentrated to give a crude sample that was used for nextreaction step without purification. LCMS (ES, m/z): 548.1 [M+H]⁺.

Step 2. (2R,3R,4S,5R)-5-((((((R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphanyl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-ylphosphonate

The crude sample from Step 1 was co-evaporated with ACN (3×2 mL),re-dissolved in ACN (3 mL), and dried by adding activated 4 Å molecularsieve (150 mg).(1R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl(2-cyanoethyl) diisopropylphosphoramidite (0.843 g, 0.952 mmol) was alsoco-evaporated with ACN (3×1 mL), re-dissolved in ACN (3 mL), and driedby adding activated 4 Å molecular sieve (150 mg). After 30 min, it wasadded to the previously prepared mixture containing pyridin-1-ium(2R,3R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-ylphosphonate. The reaction mixture was used in the next reaction stepwithout purification. LCMS (ES, m/z): 1332.1 [M+H]⁺.

Step 3. (2R,3R,4S,5R)-5-((((((R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2,5-dioxabicyclo[2.2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-ylphosphonate

To a reaction mixture from Step 2 at rt was added2-hydroperoxy-2-methylpropane (0.234 g, 2.60 mmol), and it was stirredfor 40 min. It was concentrated to give a crude product, which was usedfor the next step without purification. LCMS (ES, m/z): 1347.1 [M+H]⁺.

Step 4:(2R,3R,4S,5R)-5-((((((1S,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-ylphosphonate

To the crude from Step 3 in CH₂Cl₂ (9 mL) was added water (156 mg, 8.65mmol) and 2,2-dichloroacetic acid in CH₂Cl₂ (0.6N, 10 mL, 6 mmol). Theresulting mixture was stirred at rt for 15 min. Then, triethylsilane (4mL, 0.21 mmol) was added, and the stirring was continued for 40 min.Pyridine (1232 mg, 15.57 mmol) was added. The mixture was concentrated,and the residue was purified by reverse phase (C18) chromatographyeluted with 0 to 95% ACN in aq NH₄HCO₃ (5 mM) to give the product. LCMS(ES, m/z): 1046.3 [M+H]⁺.

Step 5.(5R,7R,8R,12aR,14R,15R,15aS,18S)-18-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-10(12H)-olate2-oxide

To pyridine (20 mL) at −40° C. under Ar was added diphenylphosphorochloridate (1.28 g, 4.78 mmol) over 5 min. To the solution at−40° C. was added a solution of (2R,3R,4S,5R)-5-((((((1S,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrothiophen-3-ylphosphonate (250 mg, 0.239 mmol, co-evaporated with pyridine, 3×10 mL)in CH₂Cl₂ (20 mL) dropwise. The resulting mixture was stirred at −40° C.for 30 min. The reaction mixture was used for the next reaction stepimmediately without purification. LCMS (ES, m/z): 1028.4 [M+H]⁺.

Step 6:(5R,7R,8R,12aR,14R,15R,15aS,18S)-18-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-10(12H)-olate2-oxide 10-sulfide

To the reaction mixture at rt from Step 5 was added3H-benzo[c][1,2]dithiol-3-one (121 mg, 0.717 mmol). After stirring at25° C. for 40 min, water (431 mg, 23.9 mmol) was added. The reactionmixture was concentrated under reduced pressure, and the residue waspurified by reverse phase (C, 18) chromatography eluted with 0 to 95%ACN in aq NH₄HCO₃ (5 mM) to give the product. LCMS (ES, m/z): 1060.3[M+H]⁺. ³¹P-NMR: (121 MHz, D₂O) δ 54.91, 54.61 (m, 1P), δ −6.08, −6.48(m, 1P).

Step 7:(5R,7R,8R,12aR,14R,15R,15aS,18S)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-18-{[tert-butyl(dimethyl)silyl]oxy}hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecine-2,10(12H)-diolate2-oxide 10-sulfide

(5R,7R,8R,12aR,14R,15R,15aS,18S)-18-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-10(12H)-olate2-oxide 10-sulfide (190 mg, 0.178 mmol) was dissolved in a solution ofmethylamine in EtOH (30%, 1 mL), and the resulting solution was stirredat rt for 2 h. Then, it was concentrated to give a crude samplecontaining the product. LCMS (ES, m/z): 833.2 [M+H]⁺.

Step 8:2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-2,10,18-trihydroxy-2-oxido-10-sulfidohexahydro-14H-15,2a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one

The crude from Step 7 was suspended in pyridine (5 mL) under Ar. To themixture was added Et₃N (1864 mg, 18.42 mmol) and triethylaminetrihydrofluoride (742 mg, 4.60 mmol) dropwise. The mixture was heated to50° C. for 16 h. Then, it was concentrated, and the residue was purifiedby preparative-HPLC (XBridge Shield RP18 OBD Column, 19×150 mm) elutedwith 0 to 14% ACN in aq NH₄HCO₃ (10 mM) over 16 min to give two productsafter concentration.

Example 264:2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-2,10,18-trihydroxy-2-oxido-10-sulfidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 1): T_(R): 11.00 min. LCMS (ES, m/z): 719.0 [M+H]⁺.¹H-NMR: (300 MHz, D₂O): δ 8.13 (s, 1H), 7.94 (s, 1H), 7.64 (s, 1H),6.11-5.97 (m, 2H), 5.92-5.85 (m, 1H), 5.25-5.05 (m, 1H), 4.87 (s, 1H),4.75 (s, 2H), 4.30-4.21 (m, 1H), 4.12-3.78 (m, 4H), 3.50-3.45 (m, 1H).³¹P-NMR: (121 MHz, D₂O) δ 52.06 (s, 1P), δ −0.86 (s, 1P)

Example 265:2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-2,10,18-trihydroxy-2-oxido-10-sulfidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 2): T_(R): 12.32 min. LCMS (ES, m/z): 719.0 [M+H]⁺.¹H-NMR: (300 MHz, D₂O): δ 8.13-8.03 (m, 2H), 7.64 (s, 1H), 6.11-5.97 (m,2H), 5.92-5.90 (m, 1H), 5.20-5.15 (m, 1H), 4.87-4.75 (m, 3H), 4.33-4.20(m, 1H), 4.12-4.00 (m, 2H), 3.90-3.78 (m, 2H), 3.45-3.40 (m, 1H).31P-NMR: (121 MHz, D₂O) δ 54.88 (s, 1P), δ-0.97 (s, 1P).

Examples 266 through 272, as shown in Table 9 below, were preparedaccording to procedures analogous to those outlined for Examples 264 and265 above using the appropriate monomeric nucleosides, described asPreparations or as obtained from commercial sources.

TABLE 9 Mass Ex. Structure Name [M − H]⁻ 266

2-amino-9-[(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10,16-trihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8- methanofuro[3,2-1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one 707 267

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-2,16-dihydroxy-2,10-dioxido-10-sulfanyloctahydro- 12H-5,8-methanofuro[3,2-1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotctradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one(Diastereomer 1) 690 268

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-2,16-dihydroxy-2,10-dioxido-10-sulfanyloctahydro- 12H-5,8-methanofuro[3,2-1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one(Diastereomer 2) 690 269

5-amino-3-[(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15-fluoro-2,16-dihydroxy-2,10-dioxido-10-sulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one(Diastereomer 1) 709 270

5-amino-3-[(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15-fluoro-2,16-dihydroxy-2,10-dioxido-10-sulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one(Diastereomer 2) 709 271

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,16-dihydroxy-2,10-dioxido-10-sulfanyloctahydro- 12H-5,8-methanofuro[3,2-1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one(Diastereomer 1) 690 272

5-amino-3-[(5R,7R,8R,12aR,14R,15aS,16S)-14-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2,16-dihydroxy-2,10-dioxido-10-sulfanyloctahydro- 12H-5,8-methanofuro[3,2-1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one(Diastereomer 2) 690

Examples 273 and 274:2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H1-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 1) and2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 2)

Step 1.(2R,3,4R,5R)-5-((((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate

To a mixture containing the crude(2R,3S,4R,5R)-5-((((((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphanyl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate (product of Step 1, Examples 244-247, crude, assumed 0.75mmol) was added tert-butyl hydroperoxide in decane (5.5M, 0.48 mL, 2.6mmol) dropwise, and the mixture was stirred at rt for 1 h. Then, it wascooled to 0° C., and a solution of Na₂S₂O₃ (553 mg) in water (2 mL) wasadded slowly. The mixture was stirred at rt for 5 min and then,concentrated to give the product. LCMS (ES, m/z): 1208.5 [M−H]⁻.

Step 2:(2R,3S,4R,5R)-5-((((((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate

To a solution of the crude from Step 1 in CH₂Cl₂ (10 mL) was added water(130 mg, 7.5 mmol) and 2,2-dichloroacetic acid (0.77 g, 6 mmol) inCH₂Cl₂ (10 mL). After 20 min, triethylsilane (20 mL) was added, andstirring was continued for 2 h. Pyridine (1 mL) was added, and thereaction mixture was concentrated. The residue was purified by reversephase (C18) chromatography eluted with 0 to 95% ACN in aq NH₄HCO₃(0.04%) to give the product. LCMS (ES, m/z): 908.2 [M+H]⁺. ¹H-NMR: (400MHz, CD₃OD) δ 8.76 (d, J=7.7 Hz, 2H), 8.65 (s, 1H), 8.56 (s, 1H),8.21-8.08 (m, 6H), 7.69 (t, J=7.4 Hz, 2H), 7.64-7.50 (m, 5H), 6.67 (ddd,J=32.7, 14.9, 4.4 Hz, 2H), 6.21-6.10 (m, 2H), 5.99-5.91 (m, 1H),5.64-5.57 (m, 1H), 5.56-5.45 (m, 3H), 5.38 (s, 3H), 4.71-4.54 (m, 6H),4.47-4.23 (m, 5H), 3.92 (d, J=3.8 Hz, 1H), 3.89-3.80 (m, 2H), 2.94 (dt,J=31.1, 5.9 Hz, 4H), 2.83-2.74 (m, 2H), 1.36-1.19 (m, 13H), 1.12 (s,2H). ³¹P-NMR: (162 MHz, CD₃OD) δ 2.55, −1.33, −3.09, −3.11, −155.89.

Step 3.(5R,7R,8S,12aR,14R,15S,15aR,16R)-2-(2-cyanoethoxy)-15,16-difluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate2-oxide

To pyridine (50 mL) under Ar was added diphenyl chlorophosphate (2.66 g,9.92 mmol). It was cooled at −40° C., and then, a solution of (2R,3S,4R,5R)-5-((((((2R,3R,4S,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluoro-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylphosphonate (450 mg, 496 mmol, co-evaporated with pyridine 3×5 mL) inCH₂Cl₂ (50 mL) was added dropwise over 20 min. The resulting mixture wasstirred at −40° C. for 20 min. The reaction mixture was immediately usedin the next step without purification. LCMS (ES, m/z): 891.1 [M+H]⁺.

Step 4:(5R,7R,8S,12aR,14R,15S,15aR,16R)-2-(2-cyanoethoxy)-15,16-difluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate2-oxide 10-sulfide

To the reaction mixture of Step 3 at −40° C. was added3H-benzo[c][1,2]dithiol-3-one (32.76 mg, 0.195 mmol) and water (125 mg,0.744 mmol). After stirring at rt for 40 min, the reaction mixture wasconcentrated under reduced pressure. The residue was purified by reversephase (C18) chromatography eluted with 0 to 95% ACN in aq NH₄HCO₃(0.04%) to give the product. LCMS (ES, m/z): 922.1 [M+H]⁺. ¹H-NMR: (400MHz, CD₃OD) δ 8.81-8.73 (m, 2H), 8.63-8.48 (m, 2H), 8.24 (s, 1H),8.21-8.10 (m, 5H), 7.73-7.56 (m, 7H), 6.83-6.72 (m, 2H), 6.28 (d, J=8.6Hz, 1H), 6.14 (d, J=8.5 Hz, 1H), 5.83-5.56 (m, 4H), 5.49 (d, J=11.1 Hz,1H), 4.76-4.46 (m, 11H), 4.37 (s, 1H), 4.29 (d, J=6.8 Hz, 1H), 4.18 (d,J=10.2 Hz, 1H), 3.04 (dd, J=6.5, 5.1 Hz, 4H), 2.83-2.68 (m, 1H), 2.06(s, 2H), 1.23 (dd, J=22.5, 6.9 Hz, 9H), 1.14-1.05 (m, 3H). ³¹P-NMR: (162MHz, CD₃OD) δ62.41, 56.84, 56.29, −3.14, −3.35, −4.77, −5.06, −60.84.

Step 5:2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomers 1 and 2)

The(5R,7R,8S,12aR,14R,15S,15aR,16R)-2-(2-cyanoethoxy)-15,16-difluoro-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-olate2-oxide 10-sulfide (380 mg, 0.377 mmol) was dissolved in a solution ofMeNH₂ in EtOH (30%, 30 mL), and the resulting solution was stirred at rtfor 2 h. Then, it was concentrated, and the residue was purified byPrep-HPLC (XBridge Shield RP18 OBD Column, 19×150 mm) eluted with 4 to10% ACN in aq NH₄HCO₃ (10 mM) over 20 min.

Example 273: 2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 1): T_(R)=10.10 min. LCMS (ES, m/z): 693.0 [M−H]⁻. ¹H-NMR:(400 MHz, CDCl₃) δ 10.84 (d, J=2.4 Hz, 1H), 10.72 (s, 1H), 10.59 (d,J=3.9 Hz, 1H), 9.01 (dd, J=19.4, 3.0 Hz, 1H), 8.56 (d, J=8.5 Hz, 1H),8.14-7.96 (m, 2H), 7.93-7.82 (m, 1H), 7.73 (dd, J=12.0, 6.5 Hz, 2H),7.31-7.19 (m, 2H), 7.15 (d, J=2.8 Hz, 1H), 6.94-6.74 (m, 3H), 6.71-6.53(m, 2H). ³¹P-NMR: (162 MHz, D₂O): δ 62.301 (s, 1P), 0.976 (s, 1P).

Example 274: 2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 2): T_(R): 14.27 min. LCMS (ES, m/z): 692.9 [M−H]⁻.¹H-NMR: (400 MHz, DMSO-d6) δ 8.31 (d, J=11.0 Hz, 2H), 8.18 (s, 1H), 6.40(dd, J=22.6, 2.6 Hz, 1H), 5.88 (d, J=8.8 Hz, 1H), 5.44 (d, J=33.7 Hz,2H), 5.35-5.23 (m, 1H), 5.24-5.05 (m, 2H), 4.48-4.34 (m, 1H), 4.31 (dd,J=10.6, 4.5 Hz, 1H), 4.16-3.87 (m, 3H). ³¹P-NMR: (162 MHz, DMSO-d6): δ55.096 (s, 1P), −2.731 (s, 1P).

Examples 275 through 288, as shown in Table 10 below, were preparedaccording to procedures analogous to those outlined in Examples 274 and275 above using the appropriate monomeric nucleosides, described asPreparations or as obtained from commercial sources.

TABLE 10 Mass Ex. Structure Name [M − H] 275

2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10-dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 1) 656 276

2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(4-amino-7H-pyirolo[2,3-d]pyrimidin-7-yl)-2,10-dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one(Diastereomer 2) 656 277

2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10-dihydroxy-2-oxido-10-sulfidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 703 278

2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10-dihydroxy-2-oxido-10-sulfidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 703 279

2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-[1,2,3]triazoIo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 694 280

2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 694 281

5-amino-3-[(5S,7R,8R,12aR,14R,15S,15aR)-14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-15-fluoro-2,10-dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one 675 282

5-amino-3-[(5R,7R,8S,12aR,14R,15S,15aR,16S)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2,10-dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one 695 283

2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl)-2-hydroxy-2,10-dioxido-10-sulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotctradecin-7-yl]-1,9- dihydro-6H-purin-6-one 657284

2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6-amino-9H-purin-9-yl)-18-fluoro-2,10-dihydroxy-2-oxido-10-sulfidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one 719 285

2-amino-9-[(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,16-difluoro-2-hydroxy-2,10-dioxido-10-sulfanyloclahydro-12H-5,8-methanofuro[3,2-1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one 710 286

2-amino-9-[(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-iniidazo[4,5-b]pyridin-3-yl)-15,16-difluoro-2-hydroxy-2,10-dioxido-10-sulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 708 287

2-amino-9-[(5R,7R,8R,12aR,14R,15S,15aR,16R)-14-(7-amino-3H-imidazo[4,5-b]pyridin-3-yl)-15,16-difluoro-2-hydroxy-2,10-dioxido-10-sulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 2) 708 288

2-amino-9-[(5R,7R,8R,12aR,14R,15aR,16R)-14-(7-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-15,15,16-trifluoro-2-hydroxy-2,10-dioxido-10-sulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one 728

Example 289:2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

Step 1:N-{9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-2-(2-cyanoethoxy)-15,16-difluoro-10-hydroxy-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}benzamide

O-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl)O-((2R,3S,4R,5R)-4-fluoro-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl)O-hydrogenphosphorothioate (product of Step 3, Example 261, 100 mg, 0.124 mmol)and diisopropylammonium tetrazolide (31.8 mg, 0.186 mmol) were azeotropewith dry CH₃CN (3×10 ml) and dried under high vacuum for 30 min.

The above mixture was dissolved in DMF (1 mL) and acetonitrile (7 mL)and added 200 mg active MS 4 Å and a solution of 2-cyanoethylN,N,N′,N′-tetraisopropylphosphorodiamidite (51.1 mg, 0.161 mmol) in 1 mldry CH₃CN. The resulting mixture was stirred at rt for 30 min, followedby addition of 1H-tetrazole (43.4 mg, 0.620 mmol). The reaction stayedat rt for 1 h and added tert-butyl hydroperoxide (5.0M in Decane) (0.074mL, 0.372 mmol). The stirring continued for 1 h and concentrated invacuo. The residue was purified by column chromatography on silica gel,eluting with 0-8% MeOH/DCM to give the desired product. LCMS (ES, m/z):922 [M+H]⁻

Step 2:2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

N-{9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-2-(2-cyanoethoxy)-15,16-difluoro-10-hydroxy-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}benzamide(90 mg, 0.098 mmol) and ammonia (7.0M in MeOH) (2 ml, 14.00 mmol) weresealed in a microwave tube. The mixture was heated to 50° C. and stirredfor 4 h. The reaction mixture was concentrated, and purified usingmass-directed reverse phase HPLC (X-Bridge BEH 150 Prep C18) using agradient solvent system with MeCN and 100 mM aqueous triethylammoniumacetate to give three desired diastereomers. Lyophilization of theproduct fractions furnished2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2,10-dihydroxy-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one.LCMS (ES, m/z): 693 [M−H]⁻. ¹H NMR (H2O-d2, 500 MHz): δ_(H) 8.23 (2H, d,J=5.2 Hz), 7.81 (1H, s), 6.42 (1H, d, J=14.2 Hz), 5.99 (1H, d, J=8.6Hz), 5.70 (1H, m), 5.40 (2H, m), 5.20 (1H, m), 4.62 (2H, m), 4.54 (1H,s), 4.15-4.30 (3H, m). ³¹P NMR: (H₂O-d₂, 202 MHz): δ −1.6, 52.1.

Example 290:2-amino-9-[1(2R,5S,7R,8R,10R,12aR,14R,15R,15aS)-15-hydroxy-2,10-dioxido-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one

To(2R,5S,7R,8R,10R,12aR,14R,15R,15aS)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-15-hydroxyoctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate)2,10-dioxide (Example 86, 5.0 mg, 0.0071 mmol) were added sodiumphosphate buffer (pH 6.8, 50 mM, 0.5 mL) and adenosine monophosphatedeaminase (3 mg). The reaction mixture was left to stir overnight,filtered, and purified by reverse phase HPLC (1-10% MeCN in aq NH₄HCO₃(100 mM)) to afford the title compound as a diammonium salt. LCMS (ES,m/z): 690 [M−H]⁻. ¹H NMR (500 MHz, D₂O): δ 8.30 (s, 1H), 8.20 (s, 1H),7.95 (s, 1H), 6.19 (s, 1H), 5.82 (d, J=6.0 Hz, 1H), 5.64 (m, 1H), 5.15(m, 1H), 4.86 (d, J=4.5 Hz, 1H), 4.60 (m, 1H), 4.48-4.53 (m, 2H), 4.38(m, 1H), 4.11 (dd, J=11.5, 4.0 Hz, 1H), 4.05 (m, 1H), 2.63 (m, 1H), 2.52(m, 1H). ³¹P NMR: (202 MHz, D₂O): δ 55.78 (s), 52.44 (s).

Examples 291 through 348 as shown in Table 11 below, were preparedaccording to procedures analogous to those outlined in Example 290above, from the starting compound (“St. Cmpd.”) indicated.

TABLE 11 Mass St. Ex. Structure Name [M − H]⁻ Cmpd. 291

9-[(5R,7R,8R,12aR,14R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-2,10,16-trihydroxy- 2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-/][1,3,6,9,11,2,10]pentaoxadiphosphacyclotctradecin-14-yl]- 1,9-dihydro-6H-purin-6-one 6423 292

9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-7 (6-amino-9H-purin-9-yl)-15-fluoro-2,10,16-trihydroxy-2,10- dioxidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]- 1,9-dihydro-6H-purin-6-one 6604 293

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fIuoro-2,10,16-trihydroxy-2,10-dioxido-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)octahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6- one 676 2 294

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR)-15-fluoro-2,10-dihydroxy-2,10-dioxido-14- (6-oxo-1,6-dihydro-9H-purin-9-yl)octahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6- one 660 21 295

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-2,10,16-trihydroxy-2,10-dioxido-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)octahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6- one 676 7 296

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-2,10,16-trihydroxy-14-(6-oxo-1,6- dihydro-9H-purin-9-yl)-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one(Diastereomer 1) 708 90 297

2-amino-9- [(2R,5R,7R,8R,10R,12aR,14R,15S,15aR,16R)-15-fluoro-2,10,16-trihydroxy-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10- disulfidooctahydro-12H-5,8-methanofuro[3,2-l)[1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one(Diastereomer 2) 708 89 298

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-2,10,16-trihydroxy-14-(6-oxo-1,6- dihydro-9H-purin-9-yl)-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one(Diastereomer 3) 708 91 299

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-2,10,16-trihydroxy-14-(6-oxo-1,6- dihydro-9H-purin-9-yl)-2,10-disulfidooctahydro-12H-5,8- methanofurol]3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one(Diastereomer 4) 708 92 300

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-16-fluoro-2,10,15-trihydroxy-14-(6-hydroxy-9H-purin-9-yl)-2,10-dioxidooctahydro- 12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6- one 676 5 301

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-2,10,15-trihydroxy-14-(6-hydroxy-9H- purin-9-yl)-16-methoxy-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 68813 302

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-16-azido-2,10,15-trihydroxy-14-(6-hydroxy-9H-purin-9-yl)-2,10-dioxidooctahydro- 12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6- one 699 14 303

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-difluoro-2,10-dihydroxy-10-oxido-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)-2- sulfidooctahydro-12H-5,8-methanofuro[3,2-/][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 694143 304

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-2,10,16-trihydroxy-14-(6-oxo-1,6- dihydro-9H-purin-9-yl)-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 70881 305

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-16-fluoro-2,10-dihydroxy-14-(6-oxo-1,6- dihydro-9H-purin-9-yl)-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one(Diastereomer 1) 692 120 306

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aR,16R)-16-fluoro-2,10-dihydroxy-14-(6-oxo-1,6- dihydro-9H-purin-9-yl)-2,10-disulfidooclahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one(Diastereomer 2) 692 118 307

2-amino-9-[(2R,5R,7R,8S,10R, 12aR,14R,15aS,16R)-16-fluoro-2,10-dihydroxy-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one(Diastereomer 3) 692 117 308

2-amino-9-[(2R,5R,7R,8R,10R, 12aR,14R,15S,15aR,16R)-15,16-difluoro-2,10-dihydroxy-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 710 247 309

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-15,16-difluoro-2,10-dihydroxy-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10- disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one(Diastereomer 2) 710 245 310

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-15,16-difluoro-2,10-dihydroxy-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10- disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one(Diastereomer 3) 710 246 311

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-15,16-difluoro-2,10-dihydroxy-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10- disulfidooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one(Diastereomer 4) 710 244 312

2-amino-9- [(5S,7R,8S,12aR,14R,15S,15aR,16R)-15-fluoro-2,10-dihydroxy-2,10-dioxido-14- (6-oxo-1,6-dihydro-9H-purin-9-yl)octahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6- one 660 25 313

2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-2,10-dihydroxy-2,10-dioxido-14-(6-oxo-l,6-dihydro-9H-purin-9-yl)octahydro- 12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6- one 642 27 314

2-amino-9- [(5R,7R,8S,12aR,14R,15R,15aR,16R)-15,16-difluoro-2,10-dihydroxy-2,10-dioxido-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)octahydro-12H-5,8-methanofuro [3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 67823 315

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-15,16-difluoro-2,10-dihydroxy-2,10-dioxido-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)octahydro-12H-5,8-methanofuro [3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 67826 316

2-amino-9- [(2aR,6aS,7R,8R,9aR,14R,14aS,15R)-5,7,12-trihydroxy-5,12-dioxido-8-(6-oxo-1,6-dihydro-9H-purin-9-yl)hexahydro- 6aH-2a,14-(epoxymethano)furo[3,2-d]oxeto[2,3-k][1,3,7,9,2,8] tetraoxadiphosphacyclotridecin-15(2H,3H)-yl-1,9-dihydro-6H-purin-6- one 686 17 317

2-amino-9- [(2R,5S,7R,8R,10R,12aR,14R,15S,15aR)-15-fluoro-2,10-dioxido-14-(6-oxo-1,6- dihydro-9H-purin-9-yl)-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one(Diastereomer 1) 692 130 318

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-15-fluoro-2,10-dioxido-14-(6-oxo-1,6- dihydro-9H-purin-9-yl)-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one(Diastereomer 2) 692 131 319

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16S)-2,10,15,16-tetrahydroxy-2,10-dioxido-14- (6-oxo-1,6-dihydro-9H-purin-9-yl)octahydro-12H-5,8-methanofuro[3,2- l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7- yl]-1,9-dihydro-6H-purin-6-one690 11 320

3-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)- 15-fluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-1][1,2,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one 693 133 321

8-[(5R,7R,8R,12aR,14S,15S,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-2,10- dioxidooctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl]pyrazolo[1,5-a][1,3,5]triazin-4(3H)-one 674 54 322

2-amino-9-[(5R,7R,8R,,12aR,14R, 15S,15aR,16R)-15-chloro-2,10,16-trihydroxy-2,10-dioxido-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)octahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 69215 323

2-amino-9-[(2R,5R,7R,8R,10R, 12aR,14R,15S,15aR,16S)-15-fluoro-16-hydroxy-2,10-dioxido-14-(6-oxo-1,6- dihydro-9H-purin-9-yl)-2,10-disulfanyloctahydro-12H-5,8- methanofuro(3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7- yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 1) 724 97 324

2-amino-9-[(5R,7R,8R,12aR,14R,15S, 15aR,16S)-15-fluoro-16-hydroxy-2,10-dioxido-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7- yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 2) 724 99 325

2-amino-9-[(5R,7R,8R,12aR,14R,15S, 15aR,16S)-15-fIuoro-16-hydroxy-2,10-dioxido-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7- yl]-1,9-dihydro-6H-purin-6-one(Diastereomer 3) 724 96 326

3-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-15,16-difluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 1)711 130 327

3-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-15,16-difluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 2)711 128 328

3-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-15,16-difluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5.8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 3)711 127 329

2-amino-9-[(2R,5R,7R,8S,10R, 12aR,14R,15S,15aR,16S)-15,16-difluoro-2,10-dioxido-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10-disulfanyloctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1) 710 144 330

2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-15,16-difluoro-2,10-dioxido-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)- 2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one(Diastereomer 2) 710 145 331

3-[(5R,7R,8S,12aR,14R,15S,15aR,16S)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-15,16-difluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one 711 147 332

2-amino-9- [(5R,7R,8S,12aR,14R,15aS,16R)-16-fluoro-2,10-dihydroxy-2,10-dioxido-14- (6-oxo-1,6-dihydro-9H-purin-9-yl)octahydro-l2H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6- one 660 22 333

9,9'-[(5R,7R,8S,12aR,14R,15R,15aS)- 2,10,15-trihydroxy-2,10-dioxidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7,14-diyl]bis(1,9-dihydro-6H-purin-6-one) 643 50 334

2-amino-9- [(5R,7R,8R,12aR,14R,15R,15aS,16R)-10,15,16-trihydroxy-2,10-dioxido-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)-2- sulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 69038 335

2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-15,16-difluoro-2,10-dioxido-14-(6-oxo- 1,6-dihydro-9H-purin-9-yl)-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one 710260 336

7-[(5R,7R,8S,12aR,14R,15aS,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)- 16-fluoro-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]imidazo[2,1-f][1,2,4]triazin-4(3H)-one (Diastereomer 1) 692 249 337

3-[(5R,7R,8S,12aR,14R,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-15,15,16-trifluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 1)729 165 338

3-[(5R,7R,8S,12aR,14R,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-15,15,16-trifluoro-2,10-dioxido-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 2)729 167 339

2-amino-9- [(5R,7R,8R,12aR,14R,15S,15aR,16S)-15-fluoro-2,16-dihydroxy-2,10-dioxido-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)-10- sulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7- yl]-1,9-dihydro-6H-purin-6-one708 266 340

5-amino-3- [(5R,7R,8R,12aR,14R,15S,15aR)-15-fluoro-2,10-dioxido-14-(7-oxo-6,7- dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 1)694 181 341

5-amino-3- [(5S,7R,8R,12aR,14R,15S,15aR)-15-fluoro-2,10-dioxido-14-(7-oxo-6,7- dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2,10- disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 2)694 182 342

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-15,16-difluoro-2,10-dioxido-14-(6-oxo- 1,6-dihydro-9H-purin-9-yl)-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 1)711 199 343

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-15,16-difluoro-2,10-dioxido-14-(6-oxo- 1,6-dihydro-9H-purin-9-yl)-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 2)711 197 344

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-15,16-difluoro-2,10-dioxido-14-(7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5- d]pyrimidin-3-yl)-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 1)712 200 345

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16R)-15,16-difluoro-2,10-dioxido-14-(7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5- d]pyrimidin-3-yl)-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 2)712 201 346

3-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-15,16-difluoro-2-hydroxy-2,10-dioxido-10-sulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 1)695 279 347

3-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-15,16-difluoro-2-hydroxy-2,10-dioxido-10-sulfanyloctahydro-12H-5,8-methanofuro(3,2-l)[1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one (Diastereomer 2)695 280 348

5-amino-3- [(5R,7R,8S,12aR,14R,15S,15aR,16S)-15,16-difluoro-2,10-dioxido-14-(6-oxo- 1,6-dihydro-9H-purin-9-yl)-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]triazolo[4,5- d]pyrimidin-7-one 711 179Biological Evaluation

The individual compounds described in the Examples are defined as STINGagonists by demonstrating binding to the STING protein with an EC₅₀ of20 uM or less in the STING Biochemical [³H]cGAMP Competition Assay(using either HAQ or wild type (WT) STING) and demonstrating interferonproduction with a 5% or greater luminescence induction at 30 uM in theINF-β secretion in the THP1 cell assay. The methods below describe eachof these assays.

[³H]-cGAMP Synthesis

2.3 mL of buffer solution containing 80 mM tris Cl, 200 mM MgCl₂ and 20mM NaCl followed by 0.32 mL of a 10 mM aq solution of GTP was added to aplastic 50 mL AMICON tube. A solution of [³H]ATP (21Ci/mmol, 45 mCi) in0.5 mL H₂O was then added followed by 1 mL of a 1 mg/mL solution of DNA(Herring testes activator DNA, Sigma, # D6898) and 53 uL of a 47 mMsolution of cGAS enzyme. Additional H₂O was added to bring the totalvolume to 10 mL.

The reaction was stirred for 2 h at 37° C. and then added directly to anAmicon Ultra-15 10K centrifuge tube and spun for 1 h at 4,000 g. Thecollected solution was then purified on a semi-prep Mono Q column usingthe following mobile phases:

-   -   A: 0.05M TrisCl pH 8.5 adjusted with 1M NaOH    -   B: 0.05M TrisCl, 0.5M NaCl pH 8.5 adjusted with 1M NaOH

Gradient: 100% A for 5 min followed by a linear gradient to 50:50 (A:B)over 25 min, 3 mL/min, 254 nm.

The collected product fractions were pooled and adjusted to a totalvolume of 30 mL with buffer A. A total yield of 15.5 mCi of [³H]cGAMPwas isolated at a radiochemical purity of 98.0% at a specific activityof 21.5Ci/mmol.

cGAS Enzyme

A recombinant DNA vector was chemically synthesized to express thetruncated human cGAS enzyme (residues 161-522). To aid in expression andpurification, the amino terminus contains a hexahistidine tag, SUMO tagand TEV cleavage site. The recombinant enzyme was overexpressed inROSETTA™ 2(DE3) Single Competent Cells (Novagen). Affinity purificationwas carried out using HIS-Select HF Nickel Affinity Gel (Sigma) followedby size exclusion chromatography using a Hi-Load 26/60 SUPERDEX200 prepgrade column (GE Healthcare). Fractions were pooled, concentrated, flashfrozen in liquid nitrogen and stored at −80° C. until needed forresearch applications.

Example 349: ³H-cGAMP Filtration Binding Assay (HAQ STING)

The ability of compounds to bind STING is quantified by their ability tocompete with tritiated cGAMP ligand for human STING receptor membraneusing a radioactive filter-binding assay. The binding assay employsSTING receptor obtained from Trichoplusia ni cell membranes (T. ni;Expression Systems, cat #94-002F, www.expressionsystems.com)overexpressing full-length HAQ STING prepared in-house and tritiatedcGAMP ligand also purified in-house.

The basic HAQ STING filtration assay protocol is as follows:

The compounds were serially titrated by the Hamilton STARPlus CORE in a96-well plate (Greiner, #651201) using a 1:3 ten-point dose responseformat. After compound preparation, a 2.2 ug/ml working concentration ofSTING membrane (SEQ. ID. No. 2) was prepared by diluting concentratedmembrane into assay buffer (lx PBS; Invitrogen # SH30028.02) anddouncing 7× using a manual tissue homogenizer (Wheaton, #357546). 148 uLof prepared membrane was then manually added to each well of a 96-welldeep-well polypropylene plate (Fisher Scientific, #12-566-121).Following membrane addition, 2 uL of either titrated test compound, DMSOcontrol (Sigma #276855), or cold cGAMP control (prepared in-house) wasadded to the appropriate wells using a Biomek FX. Compound and membranethen preincubated for 60 min at RT to allow compound binding toequilibrate. Following equilibration, 8 nM of [³H]c-GAMP ligand wasprepared by diluting into assay buffer, and 50 uL of this working stockwas then manually added to each well of the assay plate. Plates werethen incubated at RT for 60 min, and the contents of each assay platewere then filtered through a 96-well GF/B filter plate (PerkinElmer,#6005250) using a TomTec MachIII Cell Harvester equipped with 20 mMHEPES buffer (Fisher Scientific, # BP299500). The filter plates werethen dried at 55° C. for 30 min using a pressurized VWR oven before 30uL of Ultima GoldF scintillate was added to each well. Tritium levelsfor each reaction well were then measured using a PerkinElmer TopCountplate reader.

After normalization to controls, the percent activity for each compoundconcentration was calculated by measuring the amount of remainingradioactivity. The plot of percent activity versus the log of compoundconcentration was fit with a 4-parameter dose response equation tocalculate EC₅₀ values.

The final reaction conditions were:

Component Volume (uL) Final Concentration STING membrane 148 1.5 ug/ml³H-cGAMP 50 2.0 nM Low Control (cold cGAMP) 2 10 uM Test compound/DMSO 210 uM

Compound concentrations tested were 20.000, 637.00, 2.200, 0.740, 0.247,0.082, 0.027, 0.009, 0.003, and 0.001 μM with 1.0% residual DMSO.

Full-Length STING (HAQ) Virus Generation

STING virus was generated using an insect cell baculovirus system.Spodoptera frugiperda Sf21 cells (Kempbio, Inc.) were diluted to 5e5cells/ml in Sf-900II SFM media (LifeTechnologies #10902088) withoutantibiotics. The cell suspension was added to each well of a treated6-well plate (2 mL per well, 1e6 cells total), and the cells wereallowed to adhere for at least 30 min. Meanwhile, a 1 mL co-transfectionmix was assembled by combining 500 ng of HAQ STING[STING(1-379)R71H,G230 Å,H232R,R293Q-GG-AviTag-GS-HRV3C-HIS8/pBAC1] DNA(Genewiz custom synthesis) with 1 mL Sf-900II SFM media containing 10 μLCellfectin® II Reagent (Invitrogen #10362100) and 100 ng viral backboneBestBac 2.0, v-cath/chiA Deleted Linearized Baculovirus DNA (ExpressionSystems #91-002). The transfection mixtures were allowed to incubate for30 min. After incubation, media was gently removed from the adheredcells in the 6-well plate, the 1 mL transfection mixtures were added (1mL per well), and the plate was placed in a humidified incubator at 27°C. The following day, 1 mL Sf-900II SFM media (no antibiotics) was addedto each well of the 6-well plate. After media addition, the cells wereallowed to incubate with DNA (SEQ. ID. No. 3) at 27° C. for 5-7 days togenerate the P0 viral stock. To generate P1 viral stocks, 0.5 mL of P0viral supernatant was added to 50 mL uninfected Sf21 cells (seeded theday prior to infection at a density of 5×10⁵ cells/mL to allow for oneovernight doubling) in Sf-900II SFM media containing 5 g/mL gentamicin(Invitrogen #15710072). The infected cells were then incubated at 27° C.for 3d while shaking at 110 rpm (ATR Biotech Multitron Infors HT #AJ118). On day 3, P1 cultures were counted using a ViCell XR (BeckmanCoulter Life Sciences #383556) to confirm infection had occurred (cellsize ≥3 μm larger than uninfected cells and viability approximately85-95%). Cultures were harvested in 50 mL conical tubes and centrifugedat 2000×g for 10 min at 4° C. The P1 viral supernatants were poured offinto clean 50 ml centrifuge tubes, and the remaining P1 cell pelletswere used to generate Baculovirus Infected Insect Cells (BIICs)according to in-house validated SOP. Cryopreservation media containingSf-900II SFM media with 10% heat inactivated FBS, 10% DMSO (Sigma #D2650), and 5 μg/ml gentamicin was prepared in-house and sterilizedthrough 0.22 μM filter immediately prior to use. P1 cell pellets wereresuspended to a density of 2e7 cells/ml and aliquoted into cryovials (1mL per vial). Cryovials were placed in Mr. Frosty cell freezers O/N at−80° C. and transferred to liquid nitrogen for long term storage thefollowing day. To generate P2 viral stock, 0.5 mL of the P1 viralsupernatant was added to 50 mL uninfected Sf21 cells (seeded the dayprior to infection at a density of 5×10⁵ cells/mL to allow for oneovernight doubling) in Sf-900II SFM media containing 5 μg/mL gentamicin.These cells were incubated at 27° C. for 3d while shaking at 110 rpmbefore harvesting P2 stock with centrifugation at 2000×g for 10 min at4° C. The P2 viral supernatants were poured off and discarded, while theP2 cell pellets were used to generate P2 BIICs following the sameprotocol described above. The baculovirus generation protocol has beenvalidated to consistently produce P1/P2 BIICs with titers of 2e9 pfu/mL(2e7 cells/mL×100 pfu/cell).

Full-Length STING (HAQ) Expression

To generate STING membranes, P1/P2 BIICs were amplified overnight byadding thawed BIICs to Sf21 cells seeded at a density of 1.0×10⁶cells/mL. The volume of BIIC used to infect the culture was calculatedusing an assumed BIIC titer of 2e9 pfu/ml to achieve an MOI of 10 in theovernight amplification. After culturing overnight, the cells werecounted on a ViCell XR to confirm infection had occurred (cell size ≥3μm larger than uninfected cells and viability approximately 80-90%). Thevolume of infected Sf21 cells from the overnight amplification used toinfect the large-scale expression of Trichoplusia ni (T. ni; ExpressionSystems, cat #94-002F, www.expressionsystems.com) seeded at a density of1.0×10⁶ in cell media (ESF921 SFM containing 5 μg/mL gentamicin) atMOI=2.0 was calculated based on (100 pfu/infected Sf21 cell). The cellswere allowed to express for 48 h at 27° C. before harvesting the cellpellet, by centrifugation at 3,400×g for 10 min at 4° C. T. ni cellswere counted on a ViCell XR to confirm infection had occurred (cell size≥3 μm larger than uninfected cells and viability approximately 80-90%)prior to harvest.

Full-Length STING (HAQ) Membrane Generation

Buffer stock reagents:

-   -   1) 1 M HEPES pH 7.5, Teknova, Cat # H1035    -   2) 5 M NaCl, Sigma Aldrich, Cat # S5150-1 L    -   3) KCl, Sigma Aldrich, Cat #319309-500ML    -   4) Complete EDTA-free protease inhibitor tablets, Roche        Diagnostics, Cat #11873580001    -   5) Benzonase, Universal Nuclease, Pierce, Cat #88702

Lysis buffer [25 mM HEPES pH 7.5, 10 mM MgCl₂, 20 mM KCl, (Benzonase1:5000, Complete Protease Inhibitor tab/50 mL)] was added to the pelletof cells expressing full-length STING (HAQ) prepared above at 5 mL Lysisbuffer/g of cell pellet. The pellet was resuspended and dounced twentytimes using a Wheaton Dounce Homogenizer to disrupt the cell membrane.Homogenized lysate was then passed through the emulsiflex-C₅microfluidizer at a pressure close to 5000 PSI. The resuspended pelletwas centrifuged at 36,000 rpm (100,000×g) in a 45Ti rotor in theultra-high speed centrifuge for 45 min, 4° C. The supernatant wasremoved. The pellet then was resuspended in wash buffer [(25 mM HEPESpH7.5, 1 mM MgCl₂, 20 mM KCl, 1M NaCl (Complete Protease Inhibitortab/50 mL)] at a volume of 50 mL pellet/centrifuge tube. The pellet/washbuffer mixture was then homogenized, using a glass homogenizer on ice(20 strokes), followed by centrifugation at 36,000 rpm for 45 min at 4°C. The supernatant was removed. The wash step was repeated once more.The resulting membrane was resuspended in 20 mM HEPES pH 7.5, 500 mMNaCl, 10% glycerol, EDTA-free Protease Inhibitors (1 tablet/50 mL). Theprotein concentration was measured by Bradford assay (Bio-Rad ProteinAssay, Cat #500-0006), and protein enrichment was determined by SDS-PAGEand confirmed by Western blot. The resuspended membranes were stored at−80° C.

Full-Length HAQ STING [STING(1-379)R71H, G230 Å,H232R,R293Q-GG-AviTag-GS-HRV3C-HIS8]Amino Acid Sequence.

(SEQ. ID. No. 2) MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELHHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTADRAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCQTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFSGGGLNDIFEAQKIEWHEGSLE VLFQGPHHHHHHHHFull-length HAQ [STING(1-379)R71H, G230Å,H232R,R293Q-GG-AviTag-GS-HRV3C-HIS8/pBAC1] Plasmid DNA Sequence:

(SEQ. ID. No. 3) GGAACGGCTCCGCCCACTATTAATGAAATTAAAAATTCCAATTTTAAAAAACGCAGCAAGAGAAACATTTGTATGAAAGAATGCGTAGAAGGAAAGAAAAATGTCGTCGACATGCTGAACAACAAGATTAATATGCCTCCGTGTATAAAAAAAATATTGAACGATTTGAAAGAAAACAATGTACCGCGCGGCGGTATGTACAGGAAGAGGTTTATACTAAACTGTTACATTGCAAACGTGGTTTCGTGTGCCAAGTGTGAAAACCGATGTTTAATCAAGGCTCTGACGCATTTCTACAACCACGACTCCAAGTGTGTGGGTGAAGTCATGCATCTTTTAATCAAATCCCAAGATGTGTATAAACCACCAAACTGCCAAAAAATGAAAACTGTCGACAAGCTCTGTCCGTTTGCTGGCAACTGCAAGGGTCTCAATCCTATTTGTAATTATTGAATAATAAAACAATTATAAATGCTAAATTTGTTTTTTATTAACGATACAAACCAAACGCAACAAGAACATTTGTAGTATTATCTATAATTGAAAACGCGTAGTTATAATCGCTGAGGTAATATTTAAAATCATTTTCAAATGATTCACAGTTAATTTGCGACAATATAATTTTATTTTCACATAAACTAGACGCCTTGTCGTCTTCTTCTTCGTATTCCTTCTCTTTTTCATTTTTCTCTTCATAAAAATTAACATAGTTATTATCGTATCCATATATGTATCTATCGTATAGAGTAAATTTTTTGTTGTCATAAATATATATGTCTTTTTTAATGGGGTGTATAGTACCGCTGCGCATAGTTTTTCTGTAATTTACAACAGTGCTATTTTCTGGTAGTTCTTCGGAGTGTGTTGCTTTAATTATTAAATTTATATAATCAATGAATTTGGGATCGTCGGTTTTGTACAATATGTTGCCGGCATAGTACGCAGCTTCTTCTAGTTCAATTACACCATTTTTTAGCAGCACCGGATTAACATAACTTTCCAAAATGTTGTACGAACCGTTAAACAAAAACAGTTCACCTCCCTTTTCTATACTATTGTCTGCGAGCAGTTGTTTGTTGTTAAAAATAACAGCCATTGTAATGAGACGCACAAACTAATATCACAAACTGGAAATGTCTATCAATATATAGTTGCTGATCAGATCTGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATAGGATCCATGCCCCACTCCAGCCTGCATCCATCCATCCCGTGTCCCAGGGGTCACGGGGCCCAGAAGGCAGCCTTGGTTCTGCTGAGTGCCTGCCTGGTGACCCTTTGGGGGCTAGGAGAGCCACCAGAGCACACTCTCCGGTACCTGGTGCTCCACCTAGCCTCCCTGCAGCTGGGACTGCTGTTAAACGGGGTCTGCAGCCTGGCTGAGGAGCTGCACCACATCCACTCCAGGTACCGGGGCAGCTACTGGAGGACTGTGCGGGCCTGCCTGGGCTGCCCCCTCCGCCGTGGGGCCCTGTTGCTGCTGTCCATCTATTTCTACTACTCCCTCCCAAATGCGGTCGGCCCGCCCTTCACTTGGATGCTTGCCCTCCTGGGCCTCTCGCAGGCACTGAACATCCTCCTGGGCCTCAAGGGCCTGGCCCCAGCTGAGATCTCTGCAGTGTGTGAAAAAGGGAATTTCAACGTGGCCCATGGGCTGGCATGGTCATATTACATCGGATATCTGCGGCTGATCCTGCCAGAGCTCCAGGCCCGGATTCGAACTTACAATCAGCATTACAACAACCTGCTACGGGGTGCAGTGAGCCAGCGGCTGTATATTCTCCTCCCATTGGACTGTGGGGTGCCTGATAACCTGAGTATGGCTGACCCCAACATTCGCTTCCTGGATAAACTGCCCCAGCAGACCGCTGACCGTGCTGGCATCAAGGATCGGGTTTACAGCAACAGCATCTATGAGCTTCTGGAGAACGGGCAGCGGGCGGGCACCTGTGTCCTGGAGTACGCCACCCCCTTGCAGACTTTGTTTGCCATGTCACAATACAGTCAAGCTGGCTTTAGCCGGGAGGATAGGCTTGAGCAGGCCAAACTCTTCTGCCAGACACTTGAGGACATCCTGGCAGATGCCCCTGAGTCTCAGAACAACTGCCGCCTCATTGCCTACCAGGAACCTGCAGATGACAGCAGCTTCTCGCTGTCCCAGGAGGTTCTCCGGCACCTGCGGCAGGAGGAAAAGGAAGAGGTTACTGTGGGCAGCTTGAAGACCTCAGCGGTGCCCAGTACCTCCACGATGTCCCAAGAGCCTGAGCTCCTCATCAGTGGAATGGAAAAGCCCCTCCCTCTCCGCACGGATTTCTCTGGCGGTGGCCTGAACGACATCTTCGAAGCCCAGAAAATCGAATGGCATGAAGGCAGCCTGGAAGTGCTGTTCCAGGGCCCACACCACCATCATCACCATCACCATTAATGAGCGGCCGCACTCGAGCACCACCACCACCACCACTAACCTAGGTAGCTGAGCGCATGCAAGCTGATCCGGGTTATTAGTACATTTATTAAGCGCTAGATTCTGTGCGTTGTTGATTTACAGACAATTGTTGTACGTATTTTAATAATTCATTAAATTTATAATCTTTAGGGTGGTATGTTAGAGCGAAAATCAAATGATTTTCAGCGTCTTTATATCTGAATTTAAATATTAAATCCTCAATAGATTTGTAAAATAGGTTTCGATTAGTTTCAAACAAGGGTTGTTTTTCCGAACCGATGGCTGGACTATCTAATGGATTTTCGCTCAACGCCACAAAACTTGCCAAATCTTGTAGCAGCAATCTAGCTTTGTCGATATTCGTTTGTGTTTTGTTTTGTAATAAAGGTTCGACGTCGTTCAAAATATTATGCGCTTTTGTATTTCTTTCATCACTGTCGTTAGTGTACAATTGACTCGACGTAAACACGTTAAATAGAGCTTGGACATATTTAACATCGGGCGTGTTAGCTTTATTAGGCCGATTATCGTCGTCGTCCCAACCCTCGTCGTTAGAAGTTGCTTCCGAAGACGATTTTGCCATAGCCACACGACGCCTATTAATTGTGTCGGCTAACACGTCCGCGATCAAATTTGTAGTTGAGCTTTTTGGAATTATTTCTGATTGCGGGCGTTTTTGGGCGGGTTTCAATCTAACTGTGCCCGATTTTAATTCAGACAACACGTTAGAAAGCGATGGTGCAGGCGGTGGTAACATTTCAGACGGCAAATCTACTAATGGCGGCGGTGGTGGAGCTGATGATAAATCTACCATCGGTGGAGGCGCAGGCGGGGCTGGCGGCGGAGGCGGAGGCGGAGGTGGTGGCGGTGATGCAGACGGCGGTTTAGGCTCAAATGTCTCTTTAGGCAACACAGTCGGCACCTCAACTATTGTACTGGTTTCGGGCGCCGTTTTTGGTTTGACCGGTCTGAGACGAGTGCGATTTTTTTCGTTTCTAATAGCTTCCAACAATTGTTGTCTGTCGTCTAAAGGTGCAGCGGGTTGAGGTTCCGTCGGCATTGGTGGAGCGGGCGGCAATTCAGACATCGATGGTGGTGGTGGTGGTGGAGGCGCTGGAATGTTAGGCACGGGAGAAGGTGGTGGCGGCGGTGCCGCCGGTATAATTTGTTCTGGTTTAGTTTGTTCGCGCACGATTGTGGGCACCGGCGCAGGCGCCGCTGGCTGCACAACGGAAGGTCGTCTGCTTCGAGGCAGCGCTTGGGGTGGTGGCAATTCAATATTATAATTGGAATACAAATCGTAAAAATCTGCTATAAGCATTGTAATTTCGCTATCGTTTACCGTGCCGATATTTAACAACCGCTCAATGTAAGCAATTGTATTGTAAAGAGATTGTCTCAAGCTCGGATCGATCCCGCACGCCGATAACAAGCCTTTTCATTTTTACTACAGCATTGTAGTGGCGAGACACTTCGCTGTCGTCGAGGTTTAAACGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCA

Certain compounds of the disclosure were evaluated in HAQ STING in vitrobinding assay as described above. The following table tabulates thebiological data for these compounds as EC₅₀ values.

TABLE 12 ³H-cGAMP filtration binding assay for HAQ STING Compound EC₅₀(nM) Compound EC₅₀ (nM) Compound EC₅₀ (nM) Example 1 <1 Example 55 55.3Example 134 <1 Example 2 1.3 Example 56 89.4 Example 135 253.4 Example 3257.1 Example 57 437.9 Example 136 3.7 Example 4 22 Example 58 3.1Example 137 11.4 Example 5 1.1 Example 59 11.8 Example 138 738.8 Example6 <1 Example 60 42.5 Example 139 <1 Example 7 1.2 Example 61 18.2Example 140 12.9 Example 8 13.6 Example 66 117.1 Example 141 <1 Example9 7.2 Example 67 6.4 Example 142 6.2 Example 10 4.9 Example 68 63.8Example 143 1.05 Example 11 1.2 Example 69 <1 Example 144 <1 Example 12<1 Example 70 2 Example 145 <1 Example 13 1.5 Example 71 <1 Example 1465.6 Example 14 <1 Example 72 <1 Example 147 1.4 Example 15 378.1 Example73 834 Example 148 33.3 Example 16 12.4 Example 74 5.8 Example 149 8.4Example 17 121 Example 75 2.6 Example 150 <1 Example 18 53.9 Example 7675.4 Example 151 <1 Example 19 173 Example 77 160.5 Example 152 <1Example 20 <1 Example 78 14.6 Example 244 156.6 Example 21 1.3 Example79 103.2 Example 245 2.7 Example 22 4822 Example 80 1.3 Example 246 21.3Example 23 <1 Example 81 <1 Example 247 <1 Example 24 2.4 Example 8221.5 Example 290 2.5 Example 25 3.4 Example 83 3.7 Example 291 64.7Example 26 <1 Example 85 <1 Example 292 2.6 Example 27 1.3 Example 86 <1Example 293 1.9 Example 28 18.7 Example 87 <1 Example 294 6.6 Example 3091 Example 88 <1 Example 295 29.6 Example 31 94 Example 89 8272 Example296 4.1 Example 32 11.1 Example 90 1.7 Example 297 <1 Example 33 11.9Example 91 15.8 Example 298 52.9 Example 34 24.6 Example 92 114 Example299 1325 Example 35 7.9 Example 93 <1 Example 300 5.1 Example 36 21.5Example 94 24.6 Example 301 4.8 Example 37 619.6 Example 95 13.9 Example302 2.3 Example 38 3 Example 96 5.3 Example 303 <1 Example 69 <1 Example97 <1 Example 304 3.3 Example 70 2 Example 98 1.8 Example 305 326.2Example 71 <1 Example 99 <1 Example 306 3.6 Example 72 <1 Example 10016.7 Example 307 <1 Example 73 834 Example 101 8 Example 308 1.6 Example74 5.8 Example 102 <1 Example 309 4 Example 39 24.4 Example 117 1Example 310 27.6 Example 40 3.2 Example 118 1.4 Example 311 1595 Example41 11.8 Example 119 17.8 Example 312 122.5 Example 42 15.2 Example 12045.1 Example 313 210.6 Example 43 162.2 Example 121 <1 Example 314 <1Example 44 17.1 Example 123 5.1 Example 315 49.9 Example 45 24.8 Example124 <1 Example 316 2324 Example 46 11.7 Example 125 21.7 Example 317 <1Example 47 39.2 Example 126 9.7 Example 318 6.2 Example 48 1.2 Example127 24.4 Example 319 7.3 Example 49 30 Example 128 <1 Example 320 6.3Example 50 2.7 Example 129 88.6 Example 321 112.2 Example 51 287.1Example 130 <1 Example 322 68.8 Example 52 1.5 Example 131 2.4 Example332 8.2 Example 53 1.7 Example 132 25.9 Example 333 45.9 Example 54 <1Example 133 61.4

Example 350: ³H-cGAMP Filtration Binding Assay (WT STING)

The ability of compounds to bind STING is quantified by their ability tocompete with tritiated cGAMP ligand for human STING receptor membraneusing a radioactive filter-binding assay. The binding assay employsSTING receptor obtained from Trichoplusia ni cell membranes (T. ni;Expression Systems, cat #94-002F, www.expressionsystems.com)overexpressing full-length WT STING prepared in-house and tritiatedcGAMP ligand also purified in-house.

The basic WT STING filtration assay protocol is as follows:

16 nM of [³H]c-GAMP ligand was prepared by diluting into assay buffer,and 50 uL of this working stock was manually added to each well of theassay plate. After ligand addition, 2 uL of either titrated testcompound, DMSO control (Sigma #276855), or cold cGAMP control (preparedin-house) was added to the appropriate wells using a Biomek FX. Theserially titrated compound was prepared on a Hamilton STARPlus CORE in a96-well plate (Greiner, #651201) using a 1:3 ten-point dose responseformat. Following compound addition, a 2.2 ug/ml working concentrationof STING membrane (SEQ. ID. No. 4) was prepared by diluting concentratedmembrane into assay buffer (lx PBS; Invitrogen # SH30028.02) anddouncing 7× using a manual tissue homogenizer (Wheaton, #357546). 148 uLof this prepared membrane was then manually added to each well of a96-well deep-well polypropylene plate (Fisher Scientific, #12-566-121).Compound, ligand, and membrane then incubated for 60 min at RT beforethe contents of each assay plate were filtered through a 96-well GF/Bfilter plate (PerkinElmer, #6005250) using a TomTec MachIII CellHarvester equipped with 20 mM HEPES buffer (Fisher Scientific, #BP299500). The filter plates were then dried at 55° C. for 30 min usinga pressurized VWR oven before 30 uL of Ultima GoldF scintillate wasadded to each well. Tritium levels for each reaction well were thenmeasured using a PerkinElmer TopCount plate reader.

After normalization to controls, the percent activity for each compoundconcentration was calculated by measuring the amount of remainingradioactivity. The plot of percent activity versus the log of compoundconcentration was fit with a 4-parameter dose response equation tocalculate EC₅₀ values.

The final reaction conditions were:

Component Volume (uL) Final Concentration STING membrane 148 1.5 ug/ml³H-cGAMP 50 4.0 nM Low Control (cold cGAMP) 2 10 uM Test compound/DMSO 210 uM

Compound concentrations tested were 20.000, 637.00, 2.200, 0.740, 0.247,0.082, 0.027, 0.009, 0.003, and 0.001 μM with 1.0% residual DMSO.

Full-Length STING (WT) Virus Generation

STING virus was generated using an insect cell baculovirus system.Spodoptera frugiperda Sf21 cells (Kempbio, Inc.) were diluted to 5e5cells/ml in Sf-900II SFM media (LifeTechnologies #10902088) withoutantibiotics. The cell suspension was added to each well of a treated6-well plate (2 mL per well, 1e6 cells total), and the cells wereallowed to adhere for at least 30 min. Meanwhile, a 1 mL co-transfectionmix was assembled by combining 500 ng of WTSTING[STING(1-379)H232R-gg-AviTag-gs-HRV3C-HIS8/pBAC1] (Genewiz customsynthesis) with 1 mL Sf-900II SFM media containing 10 μL Cellfectin® IIReagent (Invitrogen #10362100) and 100 ng viral backbone BestBac 2.0,v-cath/chiA Deleted Linearized Baculovirus DNA (Expression Systems#91-002). The transfection mixtures were allowed to incubate for 30 min.After incubation, media was gently removed from the adhered cells in the6-well plate, the 1 mL transfection mixtures were added (1 mL per well),and the plate was placed in a humidified incubator at 27° C. Thefollowing day, 1 mL Sf-900II SFM media (no antibiotics) was added toeach well of the 6-well plate. After media addition, the cells wereallowed to incubate with DNA [(SEQ. ID. No. 5) and linearized viralbackbone BestBac 2.0] at 27° C. for 5-7 days to generate the P0 viralstock. To generate P1 viral stocks, 0.5 mL of P0 viral supernatant wasadded to 50 mL uninfected Sf21 cells (seeded the day prior to infectionat a density of 5×10⁵ cells/mL to allow for one overnight doubling) inSf-900II SFM media containing 5 g/mL gentamicin (Invitrogen #15710072).The infected cells were then incubated at 27° C. for 3d while shaking at110 rpm (ATR Biotech Multitron Infors HT # AJ118). On day 3, P1 cultureswere counted using a ViCell XR (Beckman Coulter Life Sciences #383556)to confirm infection had occurred (cell size ≥3 μm larger thanuninfected cells and viability approximately 85-95%). Cultures wereharvested in 50 mL conical tubes and centrifuged at 2000×g for 10 min at4° C. The P1 viral supernatants were poured off into clean 50 mlcentrifuge tubes, and the remaining P1 cell pellets were used togenerate Baculovirus Infected Insect Cells (BIICs) according to in-housevalidated SOP. Cryopreservation media containing Sf-900II SFM media with10% heat inactivated FBS, 10% DMSO (Sigma # D2650), and 5 μg/mlgentamicin was prepared in-house and sterilized through 0.22 μM filterimmediately prior to use. P1 cell pellets were resuspended to a densityof 2e7 cells/ml and aliquoted into cryovials (1 mL per vial). Cryovialswere placed in Mr. Frosty cell freezers O/N at −80° C. and transferredto liquid nitrogen for long term storage the following day. To generateP2 viral stock, 0.5 mL of the P1 viral supernatant was added to 50 mLuninfected Sf21 cells (seeded the day prior to infection at a density of5×10⁵ cells/mL to allow for one overnight doubling) in Sf-900II SFMmedia containing 5 μg/mL gentamicin. These cells were incubated at 27°C. for 3d while shaking at 110 rpm before harvesting P2 stock withcentrifugation at 2000×g for 10 min at 4° C. The P2 viral supernatantswere poured off and discarded, while the P2 cell pellets were used togenerate P2 BIICs following the same protocol described above. Thebaculovirus generation protocol has been validated to consistentlyproduce P1/P2 BIICs with titers of 2e9 pfu/mL (2e7 cells/mL×100pfu/cell).

Full-Length STING (WT) Expression

To generate STING membranes, P1/P2 BIICs were amplified overnight byadding thawed BIICs to Sf21 cells seeded at a density of 1.0×10⁶cells/mL. The volume of BIIC used to infect the culture was calculatedusing an assumed BIIC titer of 2e9 pfu/ml to achieve an MOI of 10 in theovernight amplification. After culturing overnight, the cells werecounted on a ViCell XR to confirm infection had occurred (cell size ≥3μm larger than uninfected cells and viability approximately 80-90%). Thevolume of infected Sf21 cells from the overnight amplification used toinfect the large-scale expression of Trichoplusia ni (T. ni; ExpressionSystems, cat #94-002F, www.expressionsystems.com) seeded at a density of1.0×10⁶ in cell media (ESF921 SFM containing 5 μg/mL gentamicin) atMOI=2.0 was calculated based on (100 pfu/infected Sf21 cell). The cellswere allowed to express for 48 h at 27° C. before harvesting the cellpellet, by centrifugation at 3,400×g for 10 min at 4° C. T. ni cellswere counted on a ViCell XR to confirm infection had occurred (cell size≥3 μm larger than uninfected cells and viability approximately 80-90%)prior to harvest.

Full-Length STING (WT) Membrane Generation

Buffer stock reagents:

-   -   1) 1 M HEPES pH 7.5, Teknova, Cat # H1035    -   2) 5 M NaCl, Sigma Aldrich, Cat # S5150-1 L    -   3) KCl, Sigma Aldrich, Cat #319309-500ML    -   4) Complete EDTA-free protease inhibitor tablets, Roche        Diagnostics, Cat #11873580001    -   5) Benzonase, Universal Nuclease, Pierce, Cat #88702

Lysis buffer [25 mM HEPES pH 7.5, 10 mM MgCl₂, 20 mM KCl, (Benzonase1:5000, Complete Protease Inhibitor tab/50 mL)] was added to the pelletof cells expressing full-length STING (WT) prepared above at 5 mL Lysisbuffer/g of cell pellet. The pellet was resuspended and dounced twentytimes using a Wheaton Dounce Homogenizer to disrupt the cell membrane.Homogenized lysate was then passed through the emulsiflex-C5microfluidizer at a pressure close to 5000 PSI. The resuspended pelletwas centrifuged at 36,000 rpm (100,000×g) in a 45Ti rotor in theultra-high speed centrifuge for 45 min, 4° C. The supernatant wasremoved. The pellet then was resuspended in wash buffer [(25 mM HEPES pH7.5, 1 mM MgCl₂, 20 mM KCl, 1M NaCl (Complete Protease Inhibitor tab/50mL)] at a volume of 50 mL/pellet/centrifuge tube. The pellet/Wash buffermixture was then homogenized, using a glass homogenizer on ice (20strokes), followed by centrifugation at 36,000 rpm for 45 min at 4° C.The supernatant was removed. The wash step was repeated once more. Theresulting membrane was resuspended in 20 mM HEPES pH 7.5, 500 mM NaCl,10% glycerol, EDTA-free Protease Inhibitors (1 tablet/50 mL). Theprotein concentration was measured by Bradford assay (Bio-Rad ProteinAssay, Cat #500-0006), and protein enrichment was determined by SDS-PAGEand confirmed by Western blot. The resuspended membranes were stored at−80° C.

Full-Length STING WT [STING(1-379)H232R-gg-AviTag-gs-HRV3C-HIS8] AminoAcid Sequence:

(SEQ. ID. No. 4) MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDRAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFSGGGLNDIFEAQKIEWHEGSLE VLFQGHHHHHHHHFull-Length WT STING [STING(1-379)H232R-gg-AviTag-gs-HRV3C-HIS8/pBAC1]Plasmid Sequence:

(SEQ. ID. No. 5) GGAACGGCTCCGCCCACTATTAATGAAATTAAAAATTCCAATTTTAAAAAACGCAGCAAGAGAAACATTTGTATGAAAGAATGCGTAGAAGGAAAGAAAAATGTCGTCGACATGCTGAACAACAAGATTAATATGCCTCCGTGTATAAAAAAAATATTGAACGATTTGAAAGAAAACAATGTACCGCGCGGCGGTATGTACAGGAAGAGGTTTATACTAAACTGTTACATTGCAAACGTGGTTTCGTGTGCCAAGTGTGAAAACCGATGTTTAATCAAGGCTCTGACGCATTTCTACAACCACGACTCCAAGTGTGTGGGTGAAGTCATGCATCTTTTAATCAAATCCCAAGATGTGTATAAACCACCAAACTGCCAAAAAATGAAAACTGTCGACAAGCTCTGTCCGTTTGCTGGCAACTGCAAGGGTCTCAATCCTATTTGTAATTATTGAATAATAAAACAATTATAAATGTCAAATTTGTTTTTTATTAACGATACAAACCAAACGCAACAAGAACATTTGTAGTATTATCTATAATTGAAAACGCGTAGTTATAATCGCTGAGGTAATATTTAAAATCATTTTCAAATGATTCACAGTTAATTTGCGACAATATAATTTTATTTTCACATAAACTAGACGCCTTGTCGTCTTCTTCTTCGTATTCCTTCTCTTTTTCATTTTTCTCTTCATAAAAATTAACATAGTTATTATCGTATCCATATATGTATCTATCGTATAGAGTAAATTTTTTGTTGTCATAAATATATATGTCTTTTTTAATGGGGTGTATAGTACCGCTGCGCATAGTTTTTCTGTAATTTACAACAGTGCTATTTTCTGGTAGTTCTTCGGAGTGTGTTGCTTTAATTATTAAATTTATATAATCAATGAATTTGGGATCGTCGGTTTTGTACAATATGTTGCCGGCATAGTACGCAGCTTCTTCTAGTTCAATTACACCATTTTTTAGCAGCACCGGATTAACATAACTTTCCAAAATGTTGTACGAACCGTTAAACAAAAACAGTTCACCTCCCTTTTCTATACTATTGTCTGCGAGCAGTTGTTTGTTGTTAAAAATAACAGCCATTGTAATGAGACGCACAAACTAATATCACAAACTGGAAATGTCTATCAATATATAGTTGCTGATCAGATCTGATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATAGGATCCATGCCCCACTCCAGCCTGCATCCATCCATCCCGTGTCCCAGGGGTCACGGGGCCCAGAAGGCAGCCTTGGTTCTGCTGAGTGCCTGCCTGGTGACCCTTTGGGGGCTAGGAGAGCCACCAGAGCACACTCTCCGGTACCTGGTGCTCCACCTAGCCTCCCTGCAGCTGGGACTGCTGTTAAACGGGGTCTGCAGCCTGGCTGAGGAGCTGCGCCACATCCACTCCAGGTACCGGGGCAGCTACTGGAGGACTGTGCGGGCCTGCCTGGGCTGCCCCCTCCGCCGTGGGGCCCTGTTGCTGCTGTCCATCTATTTCTACTACTCCCTCCCAAATGCGGTCGGCCCGCCCTTCACTTGGATGCTTGCCCTCCTGGGCCTCTCGCAGGCACTGAACATCCTCCTGGGCCTCAAGGGCCTGGCCCCAGCTGAGATCTCTGCAGTGTGTGAAAAAGGGAATTTCAACGTGGCCCATGGGCTGGCATGGTCATATTACATCGGATATCTGCGGCTGATCCTGCCAGAGCTCCAGGCCCGGATTCGAACTTACAATCAGCATTACAACAACCTGCTACGGGGTGCAGTGAGCCAGCGGCTGTATATTCTCCTCCCATTGGACTGTGGGGTGCCTGATAACCTGAGTATGGCTGACCCCAACATTCGCTTCCTGGATAAACTGCCCCAGCAGACCGGTGACCGTGCTGGCATCAAGGATCGGGTTTACAGCAACAGCATCTATGAGCTTCTGGAGAACGGGCAGCGGGCGGGCACCTGTGTCCTGGAGTACGCCACCCCCTTGCAGACTTTGTTTGCCATGTCACAATACAGTCAAGCTGGCTTTAGCCGGGAGGATAGGCTTGAGCAGGCCAAACTCTTCTGCCGGACACTTGAGGACATCCTGGCAGATGCCCCTGAGTCTCAGAACAACTGCCGCCTCATTGCCTACCAGGAACCTGCAGATGACAGCAGCTTCTCGCTGTCCCAGGAGGTTCTCCGGCACCTGCGGCAGGAGGAAAAGGAAGAGGTTACTGTGGGCAGCTTGAAGACCTCAGCGGTGCCCAGTACCTCCACGATGTCCCAAGAGCCTGAGCTCCTCATCAGTGGAATGGAAAAGCCCCTCCCTCTCCGCACGGATTTCTCTGGCGGTGGCCTGAACGACATCTTCGAAGCCCAGAAAATCGAATGGCATGAAGGCAGCCTGGAAGTGCTGTTCCAGGGCCCACACCACCATCATCACCATCACCATTAATGAGCGGCCGCACTCGAGCACCACCACCACCACCACTAACCTAGGTAGCTGAGCGCATGCAAGCTGATCCGGGTTATTAGTACATTTATTAAGCGCTAGATTCTGTGCGTTGTTGATTTACAGACAATTGTTGTACGTATTTTAATAATTCATTAAATTTATAATCTTTAGGGTGGTATGTTAGAGCGAAAATCAAATGATTTTCAGCGTCTTTATATCTGAATTTAAATATTAAATCCTCAATAGATTTGTAAAATAGGTTTCGATTAGTTTCAAACAAGGGTTGTTTTTCCGAACCGATGGCTGGACTATCTAATGGATTTTCGCTCAACGCCACAAAACTTGCCAAATCTTGTAGCAGCAATCTAGCTTTGTCGATATTCGTTTGTGTTTTGTTTTGTAATAAAGGTTCGACGTCGTTCAAAATATTATGCGCTTTTGTATTTCTTTCATCACTGTCGTTAGTGTACAATTGACTCGACGTAAACACGTTAAATAGAGCTTGGACATATTTAACATCGGGCGTGTTAGCTTTATTAGGCCGATTATCGTCGTCGTCCCAACCCTCGTCGTTAGAAGTTGCTTCCGAAGACGATTTTGCCATAGCCACACGACGCCTATTAATTGTGTCGGCTAACACGTCCGCGATCAAATTTGTAGTTGAGCTTTTTGGAATTATTTCTGATTGCGGGCGTTTTTGGGCGGGTTTCAATCTAACTGTGCCCGATTTTAATTCAGACAACACGTTAGAAAGCGATGGTGCAGGCGGTGGTAACATTTCAGACGGCAAATCTACTAATGGCGGCGGTGGTGGAGCTGATGATAAATCTACCATCGGTGGAGGCGCAGGCGGGGCTGGCGGCGGAGGCGGAGGCGGAGGTGGTGGCGGTGATGCAGACGGCGGTTTAGGCTCAAATGTCTCTTTAGGCAACACAGTCGGCACCTCAACTATTGTACTGGTTTCGGGCGCCGTTTTTGGTTTGACCGGTCTGAGACGAGTGCGATTTTTTTCGTTTCTAATAGCTTCCAACAATTGTTGTCTGTCGTCTAAAGGTGCAGCGGGTTGAGGTTCCGTCGGCATTGGTGGAGCGGGCGGCAATTCAGACATCGATGGTGGTGGTGGTGGTGGAGGCGCTGGAATGTTAGGCACGGGAGAAGGTGGTGGCGGCGGTGCCGCCGGTATAATTTGTTCTGGTTTAGTTTGTTCGCGCACGATTGTGGGCACCGGCGCAGGCGCCGCTGGCTGCACAACGGAAGGTCGTCTGCTTCGAGGCAGCGCTTGGGGTGGTGGCAATTCAATATTATAATTGGAATACAAATCGTAAAAATCTGCTATAAGCATTGTAATTTCGCTATCGTTTACCGTGCCGATATTTAACAACCGCTCAATGTAAGCAATTGTATTGTAAAGAGATTGTCTCAAGCTCGGATCGATCCCGCACGCCGATAACAAGCCTTTTCATTTTTACTACAGCATTGTAGTGGCGAGACACTTCGCTGTCGTCGAGGTTTAAACGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCA

Certain compounds of the disclosure were evaluated in WT STING in vitrobinding assay as described above. The following table tabulates thebiological data for these compounds as EC₅₀ values.

TABLE 13 ³H-cGAMP filtration binding assay for WT STING Compound EC₅₀(nM) Compound EC₅₀ (nM) Compound EC₅₀ (nM) Example 1 3.5 Example 139 1.2Example 226 27.8 Example 2 0.7 Example 141 <1 Example 227 260.6 Example3 574.3 Example 142 174.8 Example 228 1005 Example 4 43.5 Example 1444.6 Example 229 31% Inh @ 20 uM Example 5 1.5 Example 145 14 Example 230382.4 Example 6 2.9 Example 148 1932 Example 231 540.4 Example 7 <1Example 149 110.8 Example 232 3283 Example 8 81.4 Example 152 5.7Example 233 33.8 Example 9 12.4 Example 153 6.5 Example 234 275.2Example 10 23.2 Example 154 60 Example 235 14.9 Example 13 0.7 Example155 <1 Example 236 6963 Example 14 1.4 Example 156 <1 Example 237 307.2Example 20 1.4 Example 157 <1 Example 238 66.3 Example 21 1.4 Example158 40 Example 239 3.2 Example 25 16.1 Example 159 7.1 Example 240 73.6Example 26 3.7 Example 160 10.3 Example 241 471.8 Example 27 10 Example161 6.2 Example 242 1.1 Example 28 264.9 Example 162 100.7 Example 24326.3 Example 29 5835 Example 163 14.3 Example 247 1.1 Example 32 25Example 164 312.4 Example 248 31.7 Example 38 24.9 Example 165 2.9Example 249 <1 Example 41 505.9 Example 166 463.2 Example 250 17.3Example 42 219 Example 167 87.7 Example 251 <1 Example 43 1015 Example168 8.6 Example 252 768.4 Example 44 1363 Example 169 186.6 Example 25354.5 Example 45 104.8 Example 170 25.5 Example 254 39.7 Example 46 337Example 171 18.7 Example 255 301.9 Example 47 444.1 Example 172 105.9Example 256 93.5 Example 51 995.8 Example 176 16.2 Example 257 135.9Example 61 378.7 Example 177 7.9 Example 258 5 Example 62 2.6 Example178 37.4 Example 259 1.5 Example 63 68.8 Example 179 111.7 Example 2607.2 Example 64 61.6 Example 180 832.1 Example 261 21.8 Example 65 249.6Example 181 459.2 Example 262 57.3 Example 70 19.4 Example 182 3276Example 263 4.8 Example 71 4.4 Example 183 3180 Example 264 177.5Example 72 <1 Example 184 <1 Example 265 5473 Example 81 3.1 Example 185201.1 Example 266 1.9 Example 82 14.6 Example 186 30.2 Example 267 6267Example 84 6.3 Example 187 6.5 Example 268 77 Example 86 2 Example 18847.6 Example 269 4837 Example 89 <1 Example 189 423.9 Example 270 4383Example 90 4.4 Example 190 408.7 Example 271 12.8 Example 93 1.9 Example191 281.2 Example 272 749.6 Example 94 523.8 Example 192 3867 Example273 216.4 Example 95 64 Example 193 190.5 Example 274 2.3 Example 9657.8 Example 194 159.2 Example 275 21.8 Example 97 0.4 Example 195 1855Example 276 321.5 Example 98 151.7 Example 196 1033 Example 277 28.7Example 99 1.8 Example 197 1.2 Example 278 477.9 Example 100 552 Example198 19.1 Example 279 1235 Example 101 74.4 Example 199 72 Example 2801186 Example 84 6.3 Example 200 15 Example 281 2485 Example 86 2 Example201 649.5 Example 282 12190 Example 102 1 Example 202 813.5 Example 28317.2 Example 103 <1 Example 203 7.3 Example 284 128.3 Example 104 11.6Example 204 1233 Example 285 277.1 Example 105 3.9 Example 205 42.7Example 286 4010 Example 106 <1 Example 206 259.7 Example 287 80.2Example 107 17.9 Example 207 1.4 Example 288 24.9 Example 108 1784Example 208 23.3 Example 289 5.3 Example 109 1393 Example 209 9.5Example 308 3.3 Example 110 141.4 Example 210 28.5 Example 323 3.7Example 111 155.1 Example 211 1045 Example 324 10.7 Example 112 1279Example 212 11.8 Example 325 866.1 Example 113 44.2 Example 213 135Example 326 89.7 Example 114 3.3 Example 214 2.1 Example 327 14 Example115 67.9 Example 215 33.1 Example 328 74.4 Example 116 1624 Example 2163.4 Example 329 31.7 Example 117 1.7 Example 217 66.2 Example 330 204Example 118 1.6 Example 218 13.8 Example 331 182.9 Example 119 40Example 219 530.5 Example 334 110.5 Example 120 393.7 Example 220 29%Inh Example 335 5.2 @ 20 uM Example 122 179.2 Example 221 <1 Example 336422.2 Example 124 4 Example 222 60 Example 337 29.2 Example 126 447.4Example 223 302.5 Example 338 441.7 Example 128 19.5 Example 224 37.4Example 129 973.3 Example 225 779.3

Example 351: IFN-β Secretion in THP1 Cell Culture (5 h)

The ability of compounds to stimulate the secretion of interferon-betafrom THP1 cells was measured using a human IFN-β AlphaLISA kit (PerkinElmer, Cat. No. AL265F). The basic protocol is as follows:

A Labcyte Echo 550 acoustic dispenser was used to transfer 120 nL ofcompound dissolved in DMSO into the wells of an empty, sterile 384-wellmicroplate, (Corning, Cat. No. 3712). THP1 cells (American Type CultureCollection, Cat. No. TIB202) previously frozen in Recovery Medium (LifeTechnologies, Cat. No. 12648-010) were thawed and immediately diluted10-fold into 37° C. assay medium (RPMI 1640+L-Glutamine & phenol red,Life Technologies, Cat. No. 11875-085; 0.5% heat inactivated fetalbovine serum, Sigma Aldrich, Cat. No. F4135; 1 mM Sodium Pyruvate, LifeTechnologies, Cat. No. 11360-070; lx non-essential amino acids; LifeTechnologies, Cat. No. 11140-050). The cell viability and count wasascertained using a Beckman Coulter V-Cell XR cell counter. The cellssuspension was centrifuged at 200×g for 5 min at RT. Cells wereresuspended to a density of 0.8×10⁶/mL in 37° C. assay medium.Subsequent liquid transfers were performed using either a Matrixelectronic multichannel pipette or an Agilent Bravo Automated LiquidHandling Platform.

The assay was started by dispensing 40 μL of the previously preparedcell suspension into the wells of the plate containing compounds. After5 h incubation at 37° C., 5% CO₂ in a humidified atmosphere, the plateof cells and compounds was centrifuged at 200×g for 5 min at RT. Fromeach well, 5 μL of supernatant was transferred into corresponding wellsof a white 384-well plate (Perkin Elmer, Cat. No. 6005620). To thesesupernatant-containing wells was added 10 μL of 5× Anti-Analyte Acceptorbeads (50 μg/mL of AlphaLISA HiBlock Buffer) and incubated for 30 min atRT while shaking on an orbital plate shaker. To each well was added 10μL of 5× Biotinylated Antibody Anti-analyte (5 nM in AlphaLISA HiBlockBuffer) and incubated on an orbital plate shaker for 60 min at RT orovernight at 4° C. To each well was added 25 μL of 2×SA-Donor beads (80μg/mL in AlphaLISA HiBlock Buffer) and incubated for 30-45 min at RT inthe dark while shaking on an orbital plate shaker. The plate was thenread on a Perkin Elmer Envision (λ_(ex)=680 nm, λ_(em)=570 nm). Thepercent effect of the AlphaLISA signal at each compound concentrationwas calculated based on 30 uM cGAMP positive controls and 0.3% DMSOnegative controls. The plot of percent effect versus the log of compoundconcentration was fit with a 4-parameter concentration response equationto calculate EC₅₀ values. The test compounds were tested atconcentrations 30000, 10000, 3333, 1111, 370.4, 123.4, 41.2, 13.7, 4.6,and 1.5 nM with 0.3% residual DMSO. The control compound, cGAMP wastested at concentrations 100000, 33333, 11111, 3704, 1235, 412, 137, 46,and 15 nM with 0.3% residual DMSO.

Compounds of the disclosure were evaluated for IFN-β secretion in THP1cell culture as described above. The following table tabulates thebiological data for these compounds as percent activation relative to2′3′-cGAMP at the 30 μM concentration.

TABLE 14 IFN-β secretion in THP1 cell culture (5 h) % Effect at 30 μMCompound relative to 2′3′-cGAMP Example 1 154 Example 2 148 Example 3149 Example 4 171 Example 5 152 Example 6 114 Example 7 112 Example 8 98Example 9 143 Example 10 126 Example 11 169 Example 12 89 Example 13 39Example 14 139 Example 15 39 Example 16 57 Example 17 81 Example 18 74Example 19 49 Example 20 159 Example 21 142 Example 22 109 Example 23125 Example 24 90 Example 25 145 Example 26 150 Example 27 164 Example28 60 Example 29 25 Example 30 131 Example 31 100 Example 32 125 Example33 200 Example 34 125 Example 35 51 Example 36 120 Example 37 57 Example38 123 Example 39 74 Example 40 77 Example 41 8 Example 42 101 Example43 130 Example 44 135 Example 45 161 Example 46 143 Example 47 141Example 48 104 Example 49 74 Example 50 85 Example 51 36 Example 52 143Example 53 127 Example 54 83 Example 55 83 Example 56 64 Example 57 7Example 58 92 Example 59 76 Example 60 12 Example 61 34 Example 62 68Example 63 83 Example 64 93 Example 65 87 Example 66 54 Example 67 111Example 68 72 Example 69 31 Example 70 100 Example 71 113 Example 72 102Example 73 5 Example 74 57 Example 75 96 Example 76 39 Example 77 39Example 78 162 Example 79 145 Example 80 234 Example 81 138 Example 82111 Example 83 133 Example 84 128 Example 85 154 Example 86 137 Example87 99 Example 88 54 Example 89 119 Example 90 117 Example 91 106 Example92 62 Example 93 155 Example 94 138 Example 95 118 Example 96 67 Example97 113 Example 98 54 Example 99 135 Example 100 35 Example 101 129Example 102 132 Example 103 114 Example 104 154 Example 105 137 Example106 208 Example 107 171 Example 108 15 Example 109 151 Example 110 181Example 111 200 Example 112 33 Example 113 154 Example 114 163 Example115 177 Example 116 149 Example 117 211 Example 118 174 Example 119 151Example 120 72 Example 121 108 Example 122 75 Example 123 128 Example124 128 Example 125 138 Example 126 167 Example 127 145 Example 128 146Example 129 33 Example 130 215 Example 131 238 Example 132 150 Example133 117 Example 134 138 Example 135 43 Example 136 131 Example 137 124Example 138 51 Example 139 110 Example 140 96 Example 141 111 Example142 111 Example 143 157 Example 144 114 Example 145 141 Example 146 59Example 147 146 Example 148 13 Example 149 8 Example 150 89 Example 15157 Example 152 16 Example 153 234 Example 154 198 Example 155 139Example 156 119 Example 157 151 Example 158 123 Example 159 140 Example160 12 Example 161 130 Example 162 116 Example 163 127 Example 164 41Example 165 245 Example 166 143 Example 167 278 Example 168 164 Example169 117 Example 170 134 Example 171 142 Example 172 26 Example 173 142Example 174 5 Example 175 120 Example 176 114 Example 177 180 Example178 111 Example 179 66 Example 180 143 Example 181 178 Example 182 47Example 183 23 Example 184 108 Example 185 61 Example 186 179 Example187 134 Example 188 251 Example 189 93 Example 190 200 Example 191 516Example 192 50 Example 193 399 Example 194 258 Example 195 61 Example196 14 Example 197 237 Example 198 205 Example 199 139 Example 200 219Example 201 222 Example 202 171 Example 203 194 Example 204 101 Example205 132 Example 206 166 Example 207 189 Example 208 163 Example 209 170Example 210 178 Example 211 114 Example 212 285 Example 213 255 Example214 224 Example 215 277 Example 216 170 Example 217 70 Example 218 152Example 219 142 Example 220 6 Example 221 405 Example 222 171 Example223 11 Example 224 288 Example 225 467 Example 226 439 Example 227 290Example 228 6 Example 229 6 Example 230 116 Example 231 189 Example 23221 Example 233 254 Example 234 75 Example 235 262 Example 236 5 Example237 222 Example 238 183 Example 239 164 Example 240 141 Example 241 300Example 242 156 Example 243 165 Example 244 96 Example 245 234 Example246 217 Example 247 166 Example 248 42 Example 249 106 Example 250 6Example 251 175 Example 252 23 Example 253 73 Example 254 131 Example255 49 Example 256 149 Example 257 8 Example 258 120 Example 259 91Example 260 113 Example 261 90 Example 262 108 Example 263 114 Example264 123 Example 265 25 Example 266 168 Example 267 5 Example 268 181Example 269 109 Example 270 235 Example 271 229 Example 272 44 Example273 85 Example 274 124 Example 275 157 Example 276 45 Example 277 174Example 278 68 Example 279 160 Example 280 191 Example 281 8 Example 28239 Example 283 288 Example 284 260 Example 285 195 Example 286 16Example 287 202 Example 288 153 Example 289 58 Example 290 90 Example291 98 Example 292 114 Example 293 86 Example 294 84 Example 295 70Example 296 92 Example 297 176 Example 298 121 Example 299 5 Example 30086 Example 301 107 Example 302 103 Example 303 106 Example 304 104Example 305 50 Example 306 153 Example 307 174 Example 308 132 Example309 232 Example 310 117 Example 311 9 Example 312 90 Example 313 117Example 314 143 Example 315 92 Example 316 12 Example 317 107 Example318 150 Example 319 143 Example 320 115 Example 321 36 Example 322 41Example 323 92 Example 324 99 Example 325 10 Example 326 34 Example 327126 Example 328 99 Example 329 105 Example 330 67 Example 331 116Example 332 113 Example 333 10 Example 334 66 Example 335 60 Example 33685 Example 337 204 Example 338 219 Example 339 175 Example 340 306Example 341 7 Example 342 23 Example 343 255 Example 344 337 Example 345144 Example 346 182 Example 347 225 Example 348 8

It will be appreciated that various of the above-discussed and otherfeatures and functions, or alternatives thereof, may be desirablycombined into many other different systems or applications. Also thatvarious presently unforeseen or unanticipated alternatives,modifications, variations or improvements therein may be subsequentlymade by those skilled in the art which are also intended to beencompassed by the following claims.

What is claimed is:
 1. A method of inducing e-STING-dependent type Iinterferon production in a subject, said method comprising administeringa therapeutically effective amount of a compound selected from the groupconsisting of a compound of formula (I′) to the subject, wherein thecompound of formula (I′) is:

or a pharmaceutically acceptable salt thereof, wherein Base¹ and Base²are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3substituents R¹⁰, where each R¹⁰ is independently selected from thegroup consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃alkyl,C₃₋₆cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl),S(C₃₋₆cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂,and N(C₃₋₆ cycloalkyl)₂, Y and Y^(a) are each independently selectedfrom the group consisting of —O— and —S—; X^(a) and X^(a1) are eachindependently selected from the group consisting of O, and S; X^(b) andX^(b1) are each independently selected from the group consisting of O,and S; X^(c) and X^(c1) are each independently selected from the groupconsisting of OR⁹, SR⁹, and NR⁹R⁹; X^(d) and X^(d1) are eachindependently selected from the group consisting of O and S; R¹ and R²are each independently selected from the group consisting of H, F, Cl,Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R¹ and R^(1a) C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl,C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆alkynyl are substituted by 0 to 3 substituents selected from the groupconsisting of F, Cl, Br, I, OH, CN, and N₃; R² and R^(2a) are eachindependently selected from the group consisting of H, F, Cl, Br, I, OH,CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl,C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl,and —O—C₂-C₆ alkynyl, where said R² and R^(2a) C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, Br, I, OH, CN, and N₃; R³ is selected from the group consistingof H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R³ C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl,C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆alkynyl are substituted by 0 to 3 substituents selected from the groupconsisting of F, Cl, Br, I, OH, CN, and N₃; R⁴ and R^(4a) are eachindependently selected from the group consisting of H, F, Cl, Br, I, OH,CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl,C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl,and —O—C₂-C₆ alkynyl, where said R⁴ and R^(4a) C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, Br, I, OH, CN, and N₃; R⁵ is selected from the group consistingof H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁵ C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl,C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆alkynyl are substituted by 0 to 3 substituents selected from the groupconsisting of F, Cl, Br, I, OH, CN, NR⁹R⁹, and N₃; R⁶ and R^(6a) areeach independently selected from the group consisting of H, F, Cl, Br,I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁶ and R^(6a) C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl,C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆alkynyl are substituted by 0 to 3 substituents selected from the groupconsisting of F, Cl, Br, I, OH, CN, and N₃; R⁷ and R^(7a) are eachindependently selected from the group consisting of H, F, Cl, Br, I, OH,CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl,C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl,and —O—C₂-C₆ alkynyl, where said R⁷ and R^(7a) C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, Br, I, OH, CN, and N₃; R⁸ and R^(8a) are each independentlyselected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl,C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆alkynyl, where said R⁸ and R^(8a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3substituents selected from the group consisting of F, Cl, Br, I, OH, CN,and N₃; each R⁹ is independently selected from the group consisting ofH, C₁-C₂₀ alkyl,

where each R⁹ C₁-C₂₀ alkyl is optionally substituted by 0 to 3substituents independently selected from the group consisting of OH,—O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; optionallyR^(1a) and R³ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene,C₂-C₆ alkynylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆alkynylene, such that where R^(1a) and R³ are connected to form —O—C₁-C₆alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is boundat the R³ position; optionally R^(2a) and R³ are connected to form C₁-C₆alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene, —O—C₁-C₆ alkylene,—O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R^(2a) andR³ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or—O—C₂-C₆ alkynylene, said O is bound at the R³ position; optionally R³and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene,or —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected toform —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene,said O is bound at the R³ position; optionally R⁴ and R⁵ are connectedto form are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆alkynylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆alkynylene, such that where R⁴ and R⁵ are connected to form —O—C₁-C₆alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is boundat the R⁵ position; optionally R⁵ and R⁶ are connected to form —O—C₁-C₆alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that whereR⁵ and R⁶ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene,or —O—C₂-C₆ alkynylene, said O is bound at the R⁵ position; optionallyR⁷ and R⁸ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, orC₂-C₆ alkynylene; and optionally R^(7a) and R^(8a) are connected to formC₁-C₆ alkylene, C₂-C₆ alkenylene, or C₂-C₆ alkynylene; and with theprovisos that (a) when Y and Y^(a) are each O, X^(a) and X^(a1) are eachO, X^(b) and X^(b1) are each O, and X^(c) and X^(c1) are each OH or SH,X^(d) and X^(d1) are each O, R¹ and R^(1a) are each H, R² is H, R⁶ andR^(6a) are each H, R⁷ and R^(7a) are each H, R⁸ and R^(8a) are each H,and Base¹ and Base² are each selected from the group consisting of

then R⁵ and R³ are not both selected from the group consisting of H, Fand OH; and (b) the compound is not selected from the group consistingof


2. A method of inducing STING-dependent type I interferon production ina subject, said method comprising administering a therapeuticallyeffective amount of a pharmaceutical composition to the subject, whereinsaid pharmaceutical composition comprises (a) a compound of formula(I′); and (b) a pharmaceutically acceptable carrier: wherein thecompound of formula (I′) is:

or a pharmaceutically acceptable salt thereof, wherein Base¹ and Base²are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3substituents R¹⁰, where each R¹⁰ is independently selected from thegroup consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, andN(C₃₋₆ cycloalkyl)₂; Y and Y^(a) are each independently selected fromthe group consisting of —O— and —S—; X^(a) and X^(a1) are eachindependently selected from the group consisting of O, and S; X^(b) andX^(b1) are each independently selected from the group consisting of O,and S; X^(c) and X^(c1) are each independently selected from the groupconsisting of OR⁹, SR⁹, and NR⁹R⁹; X^(d) and X^(d1) are eachindependently selected from the group consisting of O and S; R¹ andR^(1a) are each independently selected from the group consisting of H,F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R¹ and R^(1a) C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl,C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆alkynyl are substituted by 0 to 3 substituents selected from the groupconsisting of F, Cl, Br, I, OH, CN, and N₃; R² and R^(2a) are eachindependently selected from the group consisting of H, F, Cl, Br, I, OH,CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl,C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl,and —O—C₂-C₆ alkynyl, where said R² and R^(2a) C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, Br, I, OH, CN, and N₃; R³ is selected from the group consistingof H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R³ C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl,C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆alkynyl are substituted by 0 to 3 substituents selected from the groupconsisting of F, Cl, Br, I, OH, CN, and N₃; R⁴ and R^(4a) are eachindependently selected from the group consisting of H, F, Cl, Br, I, OH,CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl,C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl,and —O—C₂-C₆ alkynyl, where said R⁴ and R^(4a) C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, Br, I, OH, CN, and N₃; R⁵ is selected from the group consistingof H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁵ C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl,C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆alkynyl are substituted by 0 to 3 substituents selected from the groupconsisting of F, Cl, Br, I, OH, CN, NR⁹R⁹, and N₃; R⁶ and R^(6a) areeach independently selected from the group consisting of H, F, Cl, Br,I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl, where said R⁶ and R^(6a) C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl,C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆alkynyl are substituted by 0 to 3 substituents selected from the groupconsisting of F, Cl, Br, I, OH, CN, and N₃; R⁷ and R^(7a) are eachindependently selected from the group consisting of H, F, Cl, Br, I, OH,CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl,C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl,and —O—C₂-C₆ alkynyl, where said R⁷ and R^(7a) C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, Br, I, OH, CN, and N₃; R⁸ and R^(8a) are each independentlyselected from the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl,C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆alkynyl, where said R⁸ and R^(8a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3substituents selected from the group consisting of F, Cl, Br, I, OH, CN,and N₃; each R⁹ is independently selected from the group consisting ofH, C₁-C₂₀ alkyl,

where each R⁹ C₁-C₂₀ alkyl is optionally substituted by 0 to 3substituents independently selected from the group consisting of OH,—O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; optionallyR^(1a) and R³ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene,C₂-C₆ alkynylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆alkynylene, such that where R^(1a) and R³ are connected to form —O—C₁-C₆alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is boundat the R³ position; optionally R^(2a) and R³ are connected to form C₁-C₆alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene, —O—C₁-C₆ alkylene,—O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R^(2a) andR³ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or—O—C₂-C₆ alkynylene, said O is bound at the R³ position; optionally R³and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene,or —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected toform —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene,said O is bound at the R³ position; optionally R⁴ and R⁵ are connectedto form are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆alkynylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆alkynylene, such that where R⁴ and R⁵ are connected to form —O—C₁-C₆alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is boundat the R⁵ position; optionally R⁵ and R⁶ are connected to form —O—C₁-C₆alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that whereR⁵ and R⁶ are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene,or —O—C₂-C₆ alkynylene, said O is bound at the R⁵ position; optionallyR⁷ and R⁸ are connected to form C₁-C₆ alkylene, C₂-C₆ alkenylene, orC₂-C₆ alkynylene; and optionally R^(7a) and R^(8a) are connected to formC₁-C₆ alkylene, C₂-C₆ alkenylene, or C₂-C₆ alkynylene; and (a) when Yand Y^(a) are each O, X^(a) and X^(a1) are each O, X^(b) and X^(b1) areeach O, and X^(c) and X^(c1) are each OH or SH, X^(d) and X^(d1) areeach O, R¹ and R^(1a) are each H, R² is H, R⁶ and R^(6a) are each H, R⁷and R^(7a) are each H, R⁸ and R^(8a) are each H, and Base¹ and Base² areeach selected from the group consisting of

then R⁵ and R³ are not both selected from the group consisting of H, Fand OH; and (b) the compound is not selected from the group consistingof


3. The method according to claim 1, wherein

where Base¹ and Base² each may be independently substituted by 0-3substituents R¹⁰, where each R¹⁰ is independently selected from thegroup consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, andN(C₃₋₆ cycloalkyl)₂; Y and Y^(a) are each independently selected fromthe group consisting of —O— and —S—; X^(a) and X^(a1) are eachindependently selected from the group consisting of O and S; X^(b) andX^(b1) are each independently selected from the group consisting of Oand S; X^(c) and X^(c1) are each independently selected from the groupconsisting of OR⁹, SR⁹, and NR⁹R⁹; X^(d) and X^(d1) are eachindependently selected from the group consisting of O and S; R¹ andR^(1a) are each H; R² and R^(2a) are each independently selected fromthe group consisting of H, F, Cl, Br, I, OH, N₃, C₁-C₆ alkyl, and C₁-C₆haloalkyl, where said R² and R^(2a) C₁-C₆ alkyl or C₁-C₆ haloalkyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, Br, I, OH, CN, and N₃; R³ is selected from the group consistingof H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, wheresaid R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3substituents selected from the group consisting of F, Cl, Br, I, OH, CN,and N₃; R⁴ and R^(4a) are each independently selected from the groupconsisting of H, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁴ andR^(4a) C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3substituents selected from the group consisting of F, Cl, Br, I, OH, CN,and N₃; R⁵ is selected from the group consisting of H, F, Cl, Br, I, OH,NH₂, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁵ C₁-C₆ alkyl orC₁-C₆ haloalkyl are substituted by 0 to 3 substituents selected from thegroup consisting of F, Cl, Br, I, OH, CN, NR⁹R⁹, and N₃; R³ and R⁵ arenot both selected from the group consisting of OH, C₁-C₆ alkylsubstituted with OH, and C₁-C₆ haloalkyl substituted with OH; R⁶ andR^(6a) are each independently selected from the group consisting of H,F, Cl, Br, I, OH, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, and C₁-C₆haloalkyl, where said R⁶ and R^(6a) C₁-C₆ alkyl or C₁-C₆ haloalkyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, Br, I, OH, CN, and N₃; R⁷ and R^(7a) are each H; R⁸ and R^(8a)are each H; each R⁹ is independently selected from the group consistingof H, C₂-C₃ alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2substituents independently selected from the group consisting of OH,—O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; optionally R³and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene,and —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) are connected toform —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene,said O is bound at the R³ position; and optionally R⁴ and R⁵ areconnected by C₁-C₆ alkylene, —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or—O—C₂-C₆ alkynylene, such that where R⁴ and R⁵ are connected to form—O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said Ois bound at the R⁵ position.
 4. The method according to claim 1, whereinthe compound of formula (I) is a compound of formula (Ib):

or a pharmaceutically acceptable salt thereof, wherein Base¹ and Base²are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3substituents R¹⁰, where each R¹⁰ is independently selected from thegroup consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, andN(C₃₋₆ cycloalkyl)₂; X^(c) and X^(c1) are each independently selectedfrom the group consisting of OR⁹, SR⁹, and NR⁹R⁹; R^(1a) is selectedfrom the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl,where said R^(1a) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl are substituted by 0 to 3substituents selected from the group consisting of F, Cl, Br, I, OH, CN,and N₃; R^(2a) is selected from the group consisting of H, F, Cl, Br, I,OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R^(2a) C₁-C₆alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituents selectedfrom the group consisting of F, Cl, Br, I, and OH; R³ is selected fromthe group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, andC₁-C₆ haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, Br, I, and OH; R⁵ is selected from the group consisting of H, F,Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where saidR⁵ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituentsselected from the group consisting of F, Cl, Br, I, and OH; R³ and R⁵are not both selected from the group consisting of OH, C₁-C₆ alkylsubstituted with OH, and C₁-C₆ haloalkyl substituted with OH; R^(6a) isselected from the group consisting of H, F, Cl, Br, I, OH, C₁-C₆ alkyl,C₂-C₆ alkenyl, and C₂-C₆ alkynyl; each R⁹ is independently selected fromthe group consisting of H, C₂-C₃ alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2substituents independently selected from the group consisting of OH,—O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; andoptionally R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene,—O—C₂-C₆ alkenylene, and —O—C₂-C₆ alkynylene, such that where R³ andR^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or—O—C₂-C₆ alkynylene, said O is bound at the R³ position.
 5. The methodaccording to claim 1, wherein the compound of formula (I′) is a compoundof formula (I′c):

or a pharmaceutically acceptable salt thereof, wherein Base¹ and Base²are each independently selected from the group consisting of

where Base¹ and Base² each may be independently substituted by 0-3substituents R¹⁰, where each R¹⁰ is independently selected from thegroup consisting of F, Cl, I, Br, OH, SH, NH₂, C₁₋₃ alkyl, C₃₋₆cycloalkyl, O(C₁₋₃ alkyl), O(C₃₋₆ cycloalkyl), S(C₁₋₃ alkyl), S(C₃₋₆cycloalkyl), NH(C₁₋₃ alkyl), NH(C₃₋₆ cycloalkyl), N(C₁₋₃ alkyl)₂, andN(C₃₋₆ cycloalkyl)₂; X^(c) and X^(c1) are each independently selectedfrom the group consisting of OR⁹, SR⁹, and NR⁹R⁹; R³ is selected fromthe group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, andC₁-C₆ haloalkyl, where said R³ C₁-C₆ alkyl or C₁-C₆ haloalkyl aresubstituted by 0 to 3 substituents selected from the group consisting ofF, Cl, Br, I, and OH; R⁴ is selected from the group consisting of H, F,Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁴C₁-C₆ alkyl or C₁-C₆ haloalkyl are substituted by 0 to 3 substituentsselected from the group consisting of F, Cl, Br, I, and OH; R⁵ isselected from the group consisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃,C₁-C₆ alkyl, and C₁-C₆ haloalkyl, where said R⁵ C₁-C₆ alkyl or C₁-C₆haloalkyl are substituted by 0 to 3 substituents selected from the groupconsisting of F, Cl, Br, I, and OH; R^(6a) is selected from the groupconsisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, and C₁-C₆haloalkyl, where said R⁶ C₁-C₆ alkyl or C₁-C₆ haloalkyl are substitutedby 0 to 3 substituents selected from the group consisting of F, Cl, Br,I, and OH; each R⁹ is independently selected from the group consistingof H, C₂-C₃ alkyl,

where each R⁹ C₂-C₃ alkyl is optionally substituted by 1 to 2substituents independently selected from the group consisting of OH,—O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; andoptionally R⁴ and R⁵ are connected by C₁-C₆ alkylene, —O—C₁-C₆ alkylene,—O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R⁴ and R⁵are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or—O—C₂-C₆ alkynylene, said O is bound at the R⁵ position.
 6. A method ofinducing STING-dependent type I interferon production in a subject, saidmethod comprising administering a therapeutically effective amount of acompound of formula (I″) to the subject, wherein the compound of formula(I″) is:

or a pharmaceutically acceptable salt thereof, wherein Base¹ and Base²are each independently selected from the group consisting of

Y is selected from the group consisting of —O— and —S—; X^(c) and X^(c1)are each independently selected from the group consisting of OR⁹ andSR⁹; X^(d) and X^(d1) are each independently selected from the groupconsisting of O and S; R^(2a) is selected from the group consisting ofH, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R³ is selected from the groupconsisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl,—O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R⁴ is selectedfrom the group consisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R⁵is selected from the group consisting of H, F, Cl, Br, I, OH, CN, NH₂,N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and—O—C₂-C₆ alkynyl; R^(6a) is selected from the group consisting of H, F,Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; each R⁹ is independentlyselected from the group consisting of H, C₁-C₂₀ alkyl,

where each R⁹ C₁-C₂₀ alkyl is optionally substituted by 0 to 3substituents independently selected from the group consisting of OH,—O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl; andoptionally R³ and R^(6a) are connected to form —O—C₁-C₆ alkylene,—O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, such that where R³ andR^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or—O—C₂-C₆ alkynylene, said O is bound at the R³ position; and with theprovisos that (a) when Y and Y^(a) are each O, X^(a) and X^(a1) are eachO, X^(b) and X^(b1) are each O, and X^(c) and X^(c1) are each OH or SH,X^(d) and X^(d1) are each O, R¹ and R^(1a) are each H, R² is H, R⁶ andR^(6a) are each H, R⁷ and R^(7a) are each H, R⁸ and R^(8a) are each H,and Base¹ and Base² are each selected from the group consisting of

then R⁵ and R³ are not both selected from the group consisting of H, Fand OH; and (b) the compound is not selected from the group consistingof

and pharmaceutically acceptable salts thereof.
 7. The method accordingto claim 6, wherein the compound of formula (I″) is a compound offormula (I″a):

or a pharmaceutically acceptable salt thereof, wherein Base¹ and Base²are each independently selected from the group consisting of

Y is selected from the group consisting of —O— and —S—; X^(c) and X^(c1)are each independently selected from the group consisting of OR⁹ andSR⁹; X^(d) and X^(d1) are each independently selected from the groupconsisting of O and S; R^(2a) is selected from the group consisting ofH, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R⁵ is selected from the groupconsisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl;R^(6a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN,N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and—O—C₂-C₆ alkynyl; and each R⁹ is independently selected from the groupconsisting of H, C₁-C₂₀ alkyl,

where each R⁹ C₁-C₂₀ alkyl is optionally substituted by 0 to 3substituents independently selected from the group consisting of OH,—O—C₁-C₂₀ alkyl, —S—C(O)C₁-C₆ alkyl, and C(O)OC₁-C₆ alkyl.
 8. The methodaccording to claim 7, wherein Base¹ and Base² are each independentlyselected from the group consisting of

Y is selected from the group consisting of —O— and —S—; X^(c) and X^(c1)are each independently selected from the group consisting of OR⁹ andSR⁹; X^(d) and X^(d1) are each independently selected from the groupconsisting of O and S; R^(2a) is F; R⁵ is selected from the groupconsisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl;R^(6a) is selected from the group consisting of H, F, Cl, Br, I, OH, CN,N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and—O—C₂-C₆ alkynyl; and each R⁹ is independently H.
 9. The methodaccording to claim 6, wherein R³ and R^(6a) are connected to form—O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, suchthat where R³ and R^(6a) are connected to form —O—C₂-C₆ alkylene,—O—C₂-C₆ alkenylene, or —O—C₂-C₆ alkynylene, said O is bound at the R³position.
 10. The method according to claim 6, wherein the compound offormula (I″) is a compound of formula (I″b):

or a pharmaceutically acceptable salt thereof, wherein Base¹ and Base²are each independently selected from the group consisting of

Y is selected from the group consisting of —O— and —S—; X^(c) and X^(c1)are each independently selected from the group consisting of OR⁹ andSR⁹; X^(d) and X^(d1) are each independently selected from the groupconsisting of O and S; R³ is selected from the group consisting of H, F,Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R⁴ is selected from the groupconsisting of H, F, Cl, Br, I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl,—O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; R⁵ is selectedfrom the group consisting of H, F, Cl, Br, I, OH, CN, NH₂, N₃, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ haloalkyl, C₂-C₆ haloalkenyl,C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl, —O—C₂-C₆ alkenyl, and —O—C₂-C₆alkynyl; R^(6a) is selected from the group consisting of H, F, Cl, Br,I, OH, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆haloalkyl, C₂-C₆ haloalkenyl, C₂-C₆ haloalkynyl, —O—C₁-C₆ alkyl,—O—C₂-C₆ alkenyl, and —O—C₂-C₆ alkynyl; each R⁹ is independently H; andR³ and R^(6a) are connected to form —O—C₁-C₆ alkylene, —O—C₂-C₆alkenylene, or —O—C₂-C₆ alkynylene, such that where R³ and R^(6a) areconnected to form —O—C₁-C₆ alkylene, —O—C₂-C₆ alkenylene, or —O—C₂-C₆alkynylene, said O is bound at the R³ position.
 11. The method accordingto claim 10, wherein Base¹ and Base² are each independently selectedfrom the group consisting of


12. The method according to claim 6, wherein at least one of Base¹ andBase² are each independently selected from the group consisting of


13. A method of inducing STING-dependent type I interferon production ina subject, said method comprising administering a therapeuticallyeffective amount of a compound to the subject, wherein the compound isselected from the group consisting of:

and pharmaceutically acceptable salts thereof.
 14. The method accordingto claim 13, wherein the compound is selected from the group consistingof:

and pharmaceutically acceptable salts thereof.
 15. The method accordingto claim 13, wherein the compound is selected from the group consistingof:

and pharmaceutically acceptable salts thereof.